Serge N. Manié

Abstract Tumour cells endure both oncogenic and environmental stresses during cancer progression. Transformed cells must meet increased demands for protein and lipid production needed for rapid proliferation and must adapt to exist in an oxygen‐ and nutrient‐deprived environment. To overcome such challenges, cancer cells exploit intrinsic adaptive mechanisms such as the unfolded protein response (UPR). The UPR is a pro‐survival mechanism triggered by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. IRE1, PERK and ATF6 are three ER anchored transmembrane receptors. Upon induction of ER stress, they signal in a coordinated fashion to re‐establish ER homoeostasis, thus aiding cell survival. Over the past decade, evidence has emerged supporting a role for the UPR in the establishment and progression of several cancers, including breast cancer, prostate cancer and glioblastoma multiforme. This review discusses our current knowledge of the UPR during oncogenesis, tumour growth, metastasis and chemoresistance.

Germline mutations in the RET proto-oncogene are responsible for two unrelated neural crest disorders: Hirschsprung disease, a congenital absence of the enteric nervous system in the hindgut, and multiple endocrine neoplasia type 2, a dominantly inherited cancer syndrome. Moreover, somatic rearrangements of RET are causally involved in the genesis of papillary thyroid carcinoma. The receptor tyrosine kinase encoded by the RET gene acts as the subunit of a multimolecular complex that binds four distinct ligands and activates a signalling network crucial for neural and kidney development. Over the past few years, a clearer picture of the mode of RET activation and of its multifaceted role during development has started to emerge. These findings, which provide new clues to the molecular mechanisms underlying RET signalling dysfunction in Hirschsprung disease, are summarized in this review.

The process of measles virus (MV) assembly and subsequent budding is thought to occur in localized regions of the plasma membrane, to favor specific incorporation of viral components, and to facilitate the exclusion of host proteins. We demonstrate that during the course of virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts). Isolated rafts could infect the cell through a membrane fusion step and thus contained all of the components required to create a functional virion. However, they could be distinguished from the mature virions with regards to density and Triton X-100 resistance behavior. We further show that raft localization of the viral internal nucleoprotein and matrix protein was independent of the envelope glycoproteins, indicating that raft membranes could provide a platform for MV assembly. Finally, at least part of the raft MV components were included in the viral particle during the budding process. Taken together, these results strongly suggest a role for raft membranes in the processes of MV assembly and budding.

The tetraspanins associate with various surface molecules and with each other to build a network of molecular interactions, the tetraspanin web. The interaction of tetraspanins with each other seems to be central for the assembly of the tetraspanin web. All tetraspanins studied, CD9, CD37, CD53, CD63, CD81, CD82 and CD151, were found to incorporate [3H]palmitate. By site-directed mutagenesis, CD9 was found to be palmitoylated at any of the four internal juxtamembrane regions. The palmitoylation of CD9 did not influence the partition in detergent-resistant membranes but contributed to the interaction with CD81 and CD53. In particular, the resistance of the CD9/CD81 interaction to EDTA, which disrupts other tetraspanin/tetraspanin interactions, was entirely dependent on palmitoylation.

By interacting with each others, the tetraspanins are thought to assemble a network of molecular interactions, the tetraspanin web. These tetraspanin/tetraspanin interactions involve in part the palmitoylation of the proteins. We show that tetraspanins interact with cholesterol as indicated by the precipitation of tetraspanin/tetraspanin complexes by digitonin, a cholesterol-precipitating reagent, and the labeling of the tetraspanins CD9, CD81 and CD82 with a photoactivatable cholesterol in vivo. Cholesterol may participate to the interaction of tetraspanins with each other since digitonin-precipitation of tetraspanins was correlated with their mutual interaction, and because these interactions were disrupted following cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) treatment, or cholesterol sequestration by saponin. A mutant CD9 molecule lacking all palmitoylation sites was not precipitated by digitonin under conditions in which wild-type CD9 was precipitated, indicating a role of palmitoylation for the interaction with cholesterol. Finally, upon ligation of tetraspanins on the surface of a lymphoid B cell line, the tyrosine phosphorylation of several proteins, including the vav nucleotide exchange factor, was inhibited when cells were pretreated with MbetaCD, and increased when they were treated with MbetaCD/cholesterol complexes. Thus, there is a physical and functional link between tetraspanins and cholesterol.

Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKβ, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its C terminus. Here we report that RAFTK is a target for β1-integrin-mediated tyrosine phosphorylation in both transformed and normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may have a significant function in integrin-mediated signaling pathways in B cells. Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKβ, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its C terminus. Here we report that RAFTK is a target for β1-integrin-mediated tyrosine phosphorylation in both transformed and normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may have a significant function in integrin-mediated signaling pathways in B cells.

As solid tumors expand, oxygen and nutrients become limiting owing to inadequate vascularization and diffusion. How malignant cells cope with this potentially lethal metabolic stress remains poorly understood. We found that glucose shortage associated with malignant progression triggers apoptosis through the endoplasmic reticulum (ER) unfolded protein response (UPR). ER stress is in part caused by reduced glucose flux through the hexosamine pathway. Deletion of the proapoptotic UPR effector CHOP in a mouse model of K-rasG12V-induced lung cancer increases tumor incidence, strongly supporting the notion that ER stress serves as a barrier to malignancy. Overcoming this barrier requires the selective attenuation of the PERK-CHOP arm of the UPR by the molecular chaperone p58IPK. Furthermore, p58IPK-mediated adaptive response enables cells to benefit from the protective features of chronic UPR. Altogether, these results show that ER stress activation and p58IPK expression control the fate of malignant cells facing glucose shortage.

Inflammasomes are caspase-1-activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11(-/-) cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.

The endoplasmic reticulum (ER)-induced unfolded protein response (UPR) is an adaptive mechanism that is activated upon accumulation of misfolded proteins in the ER and aims at restoring ER homeostasis. In the past 10 years, the UPR has emerged as an important actor in the different phases of tumor growth. The UPR is transduced by three major ER resident stress sensors, which are protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme-1 (IRE1). The signaling pathways elicited by those stress sensors have connections with metabolic pathways and with other plasma membrane receptor signaling networks. As such, the ER has an essential position as a signal integrator in the cell and is instrumental in the different phases of tumor progression. Herein, we describe and discuss the characteristics of an integrated signaling network that might condition the UPR biological outputs in a tissue- or stress-dependent manner. We discuss these issues in the context of the pathophysiological roles of UPR signaling in cancers.

Endoplasmic reticulum (ER) stress generates reactive oxygen species (ROS) that induce apoptosis if left unabated. To limit oxidative insults, the ER stress PKR-like endoplasmic reticulum Kinase (PERK) has been reported to phosphorylate and activate nuclear factor erythroid 2-related factor 2 (NRF2). Here, we uncover an alternative mechanism for PERK-mediated NRF2 regulation in human cells that does not require direct phosphorylation. We show that the activation of the PERK pathway rapidly stimulates the expression of NRF2 through activating transcription factor 4 (ATF4). In addition, NRF2 activation is late and largely driven by reactive oxygen species (ROS) generated during late protein synthesis recovery, contributing to protecting against cell death. Thus, PERK-mediated NRF2 activation encompasses a PERK-ATF4-dependent control of NRF2 expression that contributes to the NRF2 protective response engaged during ER stress-induced ROS production.

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. The purpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation and their ability to generate CD8+ T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCs undergo a maturation process similarly to the one induced by TNF-alpha or LPS, respectively. CD40 ligand (CD40L) expressed on activated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependent maturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expression down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12 and IL-1alpha/beta and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, we demonstrate that MV infection of DCs prevents the CD40L-dependent CD8+ T cell proliferation. In such DC-PBL cocultures, inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for the first time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate that MV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal role in MV-induced immunosuppression.

ABSTRACT During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305–311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate.

The tetraspanin web refers to a network of molecular interactions involving tetraspanins and other molecules. Inside the tetraspanin web, small primary complexes containing only one tetraspanin and one specific partner molecule such as CD151/alpha3beta1 integrin and CD9/CD9P-1 (FPRP) can be observed under particular conditions. Here we demonstrate that when cells are lysed with Brij97, the tetraspanins CD151 and CD9 allow and/or stabilize the interaction of their partner molecules with other tetraspanins and that their two partners associate under conditions maintaining tetraspanin/tetraspanin interactions. The tetraspanins were also found to partition into a detergent-resistant membrane environment to which the integrin alpha3beta1 was relocalized upon expression of CD151.

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following b1 integrin or B cell antigen receptor stimulation in human B cells.Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130 Cas and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK.In this report we observed that RAFTK was involved in the phosphorylation of these two proteins.While a catalytically active RAFTK was required for both p130 Cas and HEF1, phosphorylation of p130 Cas , but not of HEF1, was dependent on an intact autophosphorylation site (Tyr 402 ) on RAFTK.To determine if RAFTK phosphorylated p130 Cas and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr 402 mutant to phosphorylate purified HEF1 and p130 Cas domains.RAFTK was able to phosphorylate the substrate domains of both p130 Cas and HEF1, but only the C-terminal domain of p130 Cas .Furthermore, Tyr 402 , which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130 Cas .These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130 Cas .

The unfolded protein response (UPR) mediates the adaptation of the secretory pathway (SP) to fluctuations in cellular protein demand or to environmental variations. Recently, drug screenings have confirmed the therapeutic potential of targeting the UPR in cancer models. However, the UPR may not be the only druggable target of the SP. Moreover, recent studies have revealed other contributions of the SP to cancer development. This article does not intend to describe the well-established implication of UPR signaling pathways in cancer cell life and cell decision, but rather aims at defining the concept of 'tumor cell secretory addiction', from molecular, cellular, and therapeutic perspectives. Furthermore, the implication of UPR modulations in this context will be discussed.

Research over the past few years has highlighted the ability of the unfolded protein response (UPR) to minimize the deleterious effects of accumulated misfolded proteins under both physiological and pathological conditions. The endoplasmic reticulum (ER) adapts to endogenous and exogenous stressors by expanding its protein-folding capacity and by stimulating protective processes such as autophagy and antioxidant responses. Although it is clear that severe ER stress can elicit cell death, several recent studies have shown that low levels of ER stress may actually be beneficial to cells by eliciting an adaptive UPR that ‘preconditions’ the cell to a subsequent lethal insult; this process is called ER hormesis. The findings have important implications for the treatment of a wide variety of diseases associated with defective proteostasis, including neurodegenerative diseases, diabetes, and cancer. Here, we review the physiological and pathological functions of the ER, with a particular focus on the molecular mechanisms that lead to ER hormesis and cellular protection, and discuss the implications for disease treatment.

The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2α signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked β-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling.

The Crk-associated substrate p130^Cas (Cas) and the recently described human enhancer of filamentation 1 (HEF1) are two proteins with similar structure (64% amino acid homology), which are thought to act as "docking" molecules in intracellular signaling cascades. Both proteins contain an N-terminal Src homology (SH), three domain and a cluster of SH2 binding motifs. Here we show that ligation of either β1 integrin or B cell antigen receptor (BCR) on human tonsillar B cells and B cell lines promoted tyrosine phosphorylation of HEF1. In contrast, Cas tyrosine phosphorylation was observed in certain B cell lines but not in tonsillar B cells, indicating a more general role for HEF1 in B cell signaling. Interestingly, pretreatment of tonsillar B cells with cytochalasin B dramatically reduced both integrin- and BCR-induced HEF1 phosphorylation, suggesting that some component of the BCR-mediated signaling pathway is closely linked with a cytoskeletal reorganization. Both HEF1 and Cas were found to complex with the related adhesion focal tyrosine kinase (RAFTK), and when tyrosine phosphorylated, with the adapter molecule CrkL. In addition, the two molecules were detected in p53/56^Lyn immunoprecipitates, and Lyn kinase was found to specifically bind the C-terminal proline-rich sequence of Cas in an <i>in vitro</i> binding assay. These associations implicate HEF1 and Cas as important components in a cytoskeleton-linked signaling pathway initiated by ligation of β1 integrin or BCR on human B cells.

ABSTRACT The P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V − was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C − progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C − -infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C − was as potent as MV tag or MV V − in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.

T lymphocytes and monocytes were exposed to microgravity and activated to produce interleukin 2 and interleukin 1, respectively. When Jurkat T cells were triggered with monoclonal antibodies directed against the CD3/T cell receptor complex in the presence of THP-1 monocytes used as accessory cells, cell-to-cell contacts took place in microgravity leading to normal production of interleukin 2 and interleukin 1, as compared to ground controls. In contrast, when cells were individually stimulated by soluble substances including a protein kinase C activating phorbol ester, the production of interleukin 1 and interleukin 2 was dramatically inhibited during microgravity exposure. This result indicates that microgravity may affect the cellular target of phorbol ester.

CD44 is a marker of cancer stem-like cells and epithelial-mesenchymal transition that is overexpressed in many cancer types, including thyroid carcinoma. At extracellular and intramembranous domains, CD44 undergoes sequential metalloprotease- and γ-secretase-mediated proteolytic cleavage, releasing the intracellular protein fragment CD44-ICD, which translocates to the nucleus and activates gene transcription. Here, we show that CD44-ICD binds to the transcription factor CREB, increasing S133 phosphorylation and CREB-mediated gene transcription. CD44-ICD enhanced CREB recruitment to the cyclin D1 promoter, promoting cyclin D1 transcription and cell proliferation. Thyroid carcinoma cells harboring activated RET/PTC, RAS, or BRAF oncogenes exhibited CD44 cleavage and CD44-ICD accumulation. Chemical blockade of RET/PTC, BRAF, metalloprotease, or γ-secretase were each sufficient to blunt CD44 processing. Furthermore, thyroid cancer cell proliferation was obstructed by RNA interference-mediated knockdown of CD44 or inhibition of γ-secretase and adoptive CD44-ICD overexpression rescued cell proliferation. Together, these findings reveal a CD44-CREB signaling pathway that is needed to sustain cancer cell proliferation, potentially offering new molecular targets for therapeutic intervention in thyroid carcinoma.

The tetraspans associate with a large number of surface molecules, including a subset of beta1 integrins and, indirectly through CD19, with the complement receptor CD21. To further characterize the tetraspan complexes we have raised and selected monoclonal antibodies (mAb) for their ability to immunoprecipitate a molecule associated with CD9. A unique mAb was identified which recognizes the complement regulator CD46 (membrane cofactor protein). CD46 associated in part with several tetranspans and with all beta1 integrins that were tested (CD29/CD49a, CD29/CD49b, CD29/CD49c, CD29/CD49e, CD29/CD49f) but not with beta4 integrins. These data, together with cross-linking experiments showing the existence in living cells of CD46/integrin complexes, suggest that CD46 associates directly with beta1 integrins and indirectly with tetraspans. CD46 also acts as a receptor for measles virus; however, mAb to various integrins and tetraspans did not modify the virus fusion entry step.

We have demonstrated previously that microtubule depolymerization by colchicine in human monocytes induces selective production of interleukin-1 (IL-1) (Manié, S., Schmid-Alliana, A., Kubar, J., Ferrua, B., and Rossi, B. (1993) J. Biol. Chem. 268, 13675–13681). Here, we provide evidence that disruption of the microtubule structure rapidly triggers extracellular signal-regulated kinase (ERK) activation, whereas it was without effect on SAPK2 activity, which is commonly acknowledged to control pro-inflammatory cytokine production. This process involves the activation of the entire cascade including Ras, Raf-1, MEK1/2, ERK1, and ERK2. Activation of ERKs is followed by their nuclear translocation. Although other SAPK congeners might be activated upon microtubule depolymerization, the activation of ERK1 and ERK2 is mandatory for IL-1 production as shown by the blocking effect of PD 98059, a specific MEK1/2 inhibitor. Additionally, we provide evidence that microtubule disruption also induces the activation of c-Src and Hck activities. The importance of Src kinases in the mediation of the colchicine effect is underscored by the fact that CP 118556, a specific inhibitor of Src-like kinase, abrogates both the colchicine-induced ERK activation and IL-1 production. This is the first evidence that ERK activation is an absolute prerequisite for induction of this cytokine. Altogether, our data lend support to a model where the status of microtubule integrity controls the level of Src activities that subsequently activate the ERK kinase cascade, thus leading to IL-1 production. We have demonstrated previously that microtubule depolymerization by colchicine in human monocytes induces selective production of interleukin-1 (IL-1) (Manié, S., Schmid-Alliana, A., Kubar, J., Ferrua, B., and Rossi, B. (1993) J. Biol. Chem. 268, 13675–13681). Here, we provide evidence that disruption of the microtubule structure rapidly triggers extracellular signal-regulated kinase (ERK) activation, whereas it was without effect on SAPK2 activity, which is commonly acknowledged to control pro-inflammatory cytokine production. This process involves the activation of the entire cascade including Ras, Raf-1, MEK1/2, ERK1, and ERK2. Activation of ERKs is followed by their nuclear translocation. Although other SAPK congeners might be activated upon microtubule depolymerization, the activation of ERK1 and ERK2 is mandatory for IL-1 production as shown by the blocking effect of PD 98059, a specific MEK1/2 inhibitor. Additionally, we provide evidence that microtubule disruption also induces the activation of c-Src and Hck activities. The importance of Src kinases in the mediation of the colchicine effect is underscored by the fact that CP 118556, a specific inhibitor of Src-like kinase, abrogates both the colchicine-induced ERK activation and IL-1 production. This is the first evidence that ERK activation is an absolute prerequisite for induction of this cytokine. Altogether, our data lend support to a model where the status of microtubule integrity controls the level of Src activities that subsequently activate the ERK kinase cascade, thus leading to IL-1 production. Monocytes play an essential role in the inflammation as accessory cells for the processing and presentation of antigen to lymphocytes, but also in the inflammatory process by releasing oxygen metabolites, lysosomal enzymes, and cytokines such as tumor necrosis factor-α and interleukins (ILs) 1The abbreviations used are: IL, interleukin; NF, nuclear factor; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, microtubule-associated protein; MAPK, MAP kinase; MEK, MAP kinase kinase; PAGE, polyacrylamide gel electrophoresis; MBP, myelin basic protein. 1 and 6. The regulation of the synthesis of these cytokines is complex, and still poorly understood. Bacterial endotoxin (lipopolysaccharides), antigen-antibody complexes, phorbol esters (phorbol 12-myristate 13-acetate), and cytokines are classical monocyte-activating agents which induce the concerted production of these pro-inflammatory cytokines. Recent studies have shown that alteration of the cytoskeletal network by chemical agents also induces a dramatic and specific increase of IL-1 (1Manié S. Schmid-Alliana A. Kubar J. Ferrua B. Rossi B. J. Biol. Chem. 1993; 268: 13675-13681Abstract Full Text PDF PubMed Google Scholar, 2Allen N.N. Herzyk D.J. Wewers M.D. Am. J. Physiol. 1991; 261: L315-L321PubMed Google Scholar) and tumor necrosis factor-α (3Ding A. Porteu F. Sanchez E. Nathan C.F. J. Exp. Med. 1990; 171: 715-719Crossref PubMed Scopus (115) Google Scholar, 4Manthey C.L. Brandes M.E. Perera P.Y. Vogel S. J. Immunol. 1992; 149: 2459-2465PubMed Google Scholar) by monocytic cells. During recent years, growing evidence has accumulated showing that the cytoskeleton could intervene in the propagation of the mitogenic and activation signal (5Zigmond S.H. Curr. Opin. Cell Biol. 1996; 8: 66-73Crossref PubMed Scopus (276) Google Scholar). In this respect, many studies have focused on the actin network that acts as a dynamic structure involved in the integrin-mediated signaling cascade (6Craig S.W. Johnson R.P. Curr. Opin. Cell Biol. 1996; 8: 74-85Crossref PubMed Scopus (248) Google Scholar). Cytoplasmic microtubules represent another major element of the cytoskeleton that have been implicated in diverse processes such as cellular motility, intracellular transport, and secretion. The fact that microtubules are subject to constant remodeling, because of the dynamic instability of tubulin dimers, prompted us to consider that the microtubule network may be an important actor in the transmission of activation signals inside the cell. This idea is supported by reports showing that: (i) microtubule reorganization, occurring during differentiation of HL 60 cells, is associated to tubulin phosphorylation on tyrosine residues (7Katagiri K. Katagiri T. Kajiyama K. Yamamoto T. Yoshida T. J. Immunol. 1993; 150: 585-593PubMed Google Scholar); (ii) microtubule reorganization were also observed after cytokines and phorbol ester treatment of human umbilical vein endothelial cells (8Molony L. Armstrong L. Exp. Cell Res. 1991; 196: 40-48Crossref PubMed Scopus (45) Google Scholar); and (iii) microtubule disruption generates a signal that leads to NFκB activation (9Rosette C. Karin M. J. Cell Biol. 1995; 128: 1111-1119Crossref PubMed Scopus (264) Google Scholar). We and others have shown that microtubule-disrupting drugs are capable of generating a signal that leads to the selective induction of IL-1 synthesis in human monocytes (1Manié S. Schmid-Alliana A. Kubar J. Ferrua B. Rossi B. J. Biol. Chem. 1993; 268: 13675-13681Abstract Full Text PDF PubMed Google Scholar, 2Allen N.N. Herzyk D.J. Wewers M.D. Am. J. Physiol. 1991; 261: L315-L321PubMed Google Scholar). We demonstrate that microtubule depolymerization was without effect on SAPK2 activity, which is generally considered as a key regulating enzyme in the production of cytokines (10Han J. Lee J.-D. Bibbs L. Ulevitch R. Science. 1994; 265: 808-811Crossref PubMed Scopus (2446) Google Scholar, 11Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fischer J.E. Livi G.P. White J.R. Adam J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3181) Google Scholar). In contrast, we provide the first evidence that the colchicine-mediated microtubule disassembly can induce, by itself, the activation of the entire Ras-dependent cascade leading to ERK activation and their translocation to the nucleus. This cascade is in fact triggered by the upstream transient activation of c-Src and Hck. Owing to the selective effect of colchicine on IL-1 production, this study emphasizes the importance of the microtubule polymerization state in the control of the regulation it exerts on some Src-like kinases. Human myelomonocytic THP1 cells (ATCC) were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum (Biosepra), 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (referred to as the complete medium). Fetal calf serum was tested for the absence of endotoxin (<0.1 IU/ml, Institut J. Boy, Reims, France). Cells were maintained at 37 °C in a humidified 5% CO2atmosphere. Human peripheral blood mononuclear cells were isolated under sterile conditions from leukophoresis samples obtained from the Center de Transfusion Sanguine A. Tzanck (St. Laurent du Var, France) and treated as reported previously (1Manié S. Schmid-Alliana A. Kubar J. Ferrua B. Rossi B. J. Biol. Chem. 1993; 268: 13675-13681Abstract Full Text PDF PubMed Google Scholar). Monocytes were cultured for 48 h to allow adherence-induced transcription of IL-1 mRNA to subside. Human monocytes (5 × 105 cells/ml) were stimulated for 18 h in 0.5 ml of complete medium (48-well plates, Nunc) in the presence of effectors. IL-1β production (cell-associated and secreted forms) was assayed in the cell culture medium by using a specific IL-1β sandwich enzyme-linked immunosorbent assay as described previously (12Ferrua B. Becker P. Schaffar L. Shaw A. Fehlmann M. J. Immunol. Methods. 1988; 114: 41-48Crossref PubMed Scopus (40) Google Scholar). Human monocytes or THP1 cells (7 × 105cells/ml) were starved 16 h in RPMI 1640 medium and then harvested by a 5-min centrifugation at 1000 × g before being resuspended in RPMI-Hepes (Life Technologies, Inc.) at a concentration of 2 × 107 cells/ml. Cells (5 × 107) were treated with or without the effectors for 3 h at 37 °C. Nuclei were isolated from cells, and the run-on transcription assay was performed as described (13Doglio A. Dani C. Grimaldi P. Ailhaud G. Biochem. J. 1988; 238: 123-129Crossref Scopus (101) Google Scholar). Colchicine and lumicolchicine were obtained from Sigma. Genistein and herbimycin A were from Life Technologies, Inc. PD 98059, the microtubule-associated protein (MAP) kinase kinase (MEK) inhibitor, was purchased from New England Biolabs (Beverly, MA). CP 118556 (also named PP1) was kindly provided by S. Kadin (Pfizer Research, Groton, CT). SB 203580 was a gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Pervanadate was prepared as described previously (14Imbert V. Peyron J.-F. Farahi Far D. Mari B. Auberger P. Rossi B. Biochem. J. 1994; 297: 163-173Crossref PubMed Scopus (126) Google Scholar). Human monocytes or THP1 cells (7 × 105cells/ml) were starved 16 h in RPMI 1640 medium and harvested by centrifugation for 5 min at 1000 g before being resuspended in RPMI-Hepes (Life Technologies, Inc.) at a concentration of 2 × 107 cells/ml. Cells (107) were treated with or without the effectors for the indicated times at 37 °C and lysed in buffer (150 mm NaCl, 0.8 mm MgCl2, 5 mm EGTA, 1% Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, 15 μg/ml leupeptin, 1 μmpepstatin, 1 mm Na3VO4, and 50 mm Hepes, pH 7.5). The crude lysates were centrifuged at 18,000 × g for 20 min at 4 °C, and the supernatants were precleared with nonimmune serum prebound to protein A-Sepharose (Pharmacia Biotech Inc.) for rabbit serum or protein G-Sepharose (Santa Cruz Biotechnology, Santa Cruz, CA) for sheep serum. The precleared lysates were incubated at 4 °C for 16 h with antibodies raised against the various transduction proteins previously bound to protein A-Sepharose or to protein G-Sepharose. All antibodies were used at dilution 1/100. The Src-related kinases were immunoprecipitated with anti-c-Src antibodies (Santa Cruz Biotechnology) bound to protein G-Sepharose or with anti-Hck antibodies (gift from I. Maridonneau-Parini) bound to protein A-Sepharose. The immunopellets were washed twice with lysis buffer, followed by one wash with lysis buffer supplemented with 0.25% deoxycholate, and ultimately washed twice in Src kinase buffer (5 mm MgCl2, 5 mm MnCl2, 30 mm Hepes, pH 7.5). Samples were then resuspended in 50 μl of Src kinase buffer supplemented with 0.1 mg/ml acid-denatured enolase, which was used as an exogenous substrate. The kinase assay was started by addition of 40 μCi/ml [γ-32P]ATP (370 MBq/ml, Amersham Life Science). After addition of 25 μl of 9 × Laemmli sample buffer to stop the reaction, samples were heated to 95 °C for 3 min and analyzed by SDS-PAGE on a 10% gel under reducing conditions. Gels were exposed for autoradiography on Hyperfilm (Amersham). MAP-related kinases were immunoprecipitated with anti-SAPK2/P38, anti-ERK1, or anti-ERK2 antisera (Santa Cruz Biotechnology) bound to protein A-Sepharose. Immunopellets were washed twice with lysis buffer, twice with MAP kinase buffer (30 mm NaCl, 0.1% Nonidet P-40, 10% glycerol, 200 μm Na3VO4, 30 mm Hepes, pH 7.5), and resuspended in 50 μl of MAP kinase buffer in the presence of 75 μm ATP, 30 mm Mg acetate, and 0.2 mg/ml myelin basic protein (MBP, Sigma), which was used as an exogenous substrate. The kinase assay was initiated by addition of 100 μCi/ml [γ-32P]ATP. After addition of 25 μl of 9 × Laemmli sample buffer to stop the reaction, the samples were heated to 95 °C for 3 min. Samples were split to be analyzed by SDS-PAGE. Gels were either exposed for autoradiography using hyperfilms (Amersham) or probed with antibodies for immunoblotting experiments. Autoradiographies were scanned using a Ultro-Scan laser densitometer. MEK was immunoprecipitated from colchicine-treated cells with anti-MEK-1 antibody (Santa Cruz Biotechnology) bound to protein A-Sepharose. The immunopellets were washed twice with lysis buffer and twice with MEK kinase buffer (1 mm MnCl2, 10 mm MgCl2, 1 mm dithiothreitol, 10 mm p-nitrophenylphosphate, 30 mm Hepes, pH 7.5). In parallel, using specific antibodies, we immunoprecipitated ERK1, from unstimulated THP1 lysates, to be used as a substrate. The pellets containing MEK and ERK1, respectively, were then mixed in a final volume of 50 μl of MEK kinase buffer in the presence of 15 μm ATP. The reaction was started by addition of 100 μCi/ml [γ-32P]ATP. After addition of 25 μl of 9 × Laemmli sample buffer to stop the reaction, the samples were heated to 95 °C for 3 min. Samples were split to be analyzed by SDS-PAGE under reducing conditions, proteins were revealed either by autoradiography or by Western blotting using appropriate antibodies. Raf-1 was immunoprecipitated with anti-c-Raf-1 antibody (Santa Cruz Biotechnology) bound to protein A-Sepharose. The immunopellets were washed twice with lysis buffer and twice with MEK kinase buffer. In parallel, MEK was immunoprecipated from lysates of unstimulated THP1 with anti-MEK-1 antibodies to be used as a Raf-1 substrate. The pellets containing Raf-1 or MEK were then mixed to a final volume of 50 μl of MEK kinase buffer in the presence of 15 μm ATP. The reaction was started by addition of 100 μCi/ml [γ-32P]ATP and stopped by addition of 25 μl of 9 × Laemmli sample buffer. Samples were heated to 95 °C for 3 min and split for analysis by autoradiography or Western blotting after separation by SDS-PAGE under reducing conditions. Total cell lysates (100 μg), immunoprecipitated substrates, or nuclear proteins (100 μg) were separated by SDS-PAGE and transferred to Immobilon membrane as detailed previously (15Schmid-Antomarchi H. Benkirane M. Breittmayer V. Husson H. Ticchioni M. Devaux C. Rossi B. Eur. J. Immunol. 1996; 26: 717-720Crossref PubMed Scopus (47) Google Scholar). The blots were probed with 4G10 anti-phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY) or anti-c-Src at 1 μg/ml, or with anti-ERK1, anti-ERK2, anti-SAPK2/P38, anti-Raf-1, anti-MEK-1, or anti-Hck (Santa Cruz Biotechnology) at 0.1 μg/ml, the proteins were visualized by the Amersham ECL system and quantified by densitometric scanning. THP1 cells (7 × 105 cells/ml) were starved for 16 h in RPMI 1640 medium and subsequently labeled for 3 h at a concentration of 2 × 107 cells/ml with 1 mCi of [32P]orthophosphate (200 mCi/mmol, Amersham) in phosphate-free buffer (140 mm NaCl, 5 mm KCl, 0.8 mm MgCl2, 1.8 mmCaCl2, 10 mm glucose, 20 mm Hepes, pH 7.4). Cells (107) were stimulated or not by colchicine (1 μm) for the indicated times at 37 °C and rapidly washed with phosphate-free buffer, before being lysed in buffer (150 mm NaCl, 0.8 mm MgCl2, 5 mm EGTA, 1% Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, 15 μg/ml leupeptin, 1 μmpepstatin, 1 mm Na3VO4, and 50 mm Hepes, pH 7.5). After 20 min, NaCl, SDS, and deoxycholate were added to a final concentration of 0.5 m, 0.5% and 0.05%, respectively. The crude lysates were centrifuged at 18 000 × g for 20 min at 4 °C and the supernatants were precleared by incubation with rat nonimmune serum bound to protein G-Sepharose for 30 min. The treated lysates were then incubated at 4 °C for 2 h with anti-p21ras prebound to protein G-Sepharose (Santa Cruz Biotechnology). The immunoprecipitates were collected and washed eight times with 50 mm Hepes buffer, pH 7.4, 500 mm NaCl, 5 mm MgCl2, 0.1% Nonidet P-40, 0.005% SDS. GTP and GDP were finally eluted in 5 mm dithiothreitol, 5 mm EDTA, 0.2% SDS, 0.5 mm GTP, and 0.5 mm GDP at 68 °C for 20 min and separated on polyethyleneimine-cellulose plates (Schleicher & Schüll), developed in 1.2 m ammonium formate, 0.8m HCl. Plates were exposed for autoradiography, and the GTP/GDP ratio was determined by densitometric scanning. To elucidate the step at which colchicine controls the induction of IL-1β mRNA expression (1Manié S. Schmid-Alliana A. Kubar J. Ferrua B. Rossi B. J. Biol. Chem. 1993; 268: 13675-13681Abstract Full Text PDF PubMed Google Scholar), run-on experiments were carried out. Nascent nuclear RNA chains, biosynthetically labeled with [α-32P]UTP, were isolated from human monocytes previously stimulated for 3 h with colchicine or lumicolchicine. Labeled RNAs were then hybridized to nitrocellulose filters previously spotted with plasmids harboring either the IL-1β or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding sequence. A detectable constitutive level of IL-1β transcription was observed (Fig. 1, lane 1), but the treatment with colchicine dramatically increased the IL-1β transcription rate (Fig. 1, lane 3). Under the same conditions, lumicolchicine, the inactive analog of colchicine, was without any effect (lane 2). The slight inhibition that lumicolchicine induced on IL-1β transcription was not significant when normalized against the corresponding GAPDH spot. These results suggest that disruption of the microtubule network increased pro-IL-1β mRNAs at the level of transcription. It has been reported that microtubule-disrupting agents, such as colchicine or microtubule-stabilizing agents, e.g. taxol, could modulate tyrosine kinase activities in intact cells (4Manthey C.L. Brandes M.E. Perera P.Y. Vogel S. J. Immunol. 1992; 149: 2459-2465PubMed Google Scholar, 16Gaudry M. Roberge C. De Medicis R. Lussier A. Poubelle P.E. Naccache P.H. J. Clin. Invest. 1993; 92: 1722-1729Crossref PubMed Scopus (71) Google Scholar, 17Ding A. Sanchez E. Nathan C., F. J. Immunol. 1993; 151: 5596-5602PubMed Google Scholar). On the basis of these observations, we hypothesized that tyrosine kinase activities might participate in colchicine-induced IL-1β production. We first tested the effects of herbimycin A and genistein, two potent tyrosine kinase inhibitors, on the colchicine-induced IL-1β production in human monocytes. As shown in Fig. 2 A (upper panel), herbimycin A and genistein both inhibited colchicine-induced IL-1β production, with ID50 values of 10 nm and 3 μm, respectively. Conversely, as shown in Fig. 2 A (lower panel), the colchicine-induced IL-1β production was dramatically increased in the presence of pervanadate, a potent inhibitor of tyrosine phosphatases (14Imbert V. Peyron J.-F. Farahi Far D. Mari B. Auberger P. Rossi B. Biochem. J. 1994; 297: 163-173Crossref PubMed Scopus (126) Google Scholar) with a maximal synergizing effect at 30 μm. Similar effects of herbimycin A, genistein, and pervanadate were obtained when the production of the IL-1α protein was assayed instead of IL-1β (data not shown). It is noteworthy that herbimycin A does not prevent colchicine-induced cAMP accumulation in human monocytes (data not shown), suggesting that the colchicine-induced cAMP response, as reported previously (18Jasper J.R. Post S.R. Desai K.H. Insel P.A. Bernstein D. J. Pharmacol. Exp. Ther. 1995; 274: 937-942PubMed Google Scholar, 19Manié S. Proudfoot A. Ferrua B. Eur. Cytokine Netw. 1993; 4: 51-56PubMed Google Scholar), is regulated by a pathway distinct from that controlled by the herbimycin A-sensitive tyrosine kinase activity. In response to microtubule network disruption, the activation of a tyrosine kinase activity was confirmed by the detection of phosphotyrosine proteins following colchicine treatment. As shown in Fig. 2 B, exposure of THP1 cells to colchicine resulted in an increased tyrosine phosphorylation of five proteins with apparent molecular masses of 27, 28, 42, 55, and 60 kDa, respectively. The presence of tyrosine-phosphorylated protein(s) migrating in the 42–44-kDa range prompted us to investigate the possibility that MAPK congeners were activated upon microtubule disruption. We focused first our attention on SAPK2/p38 activity, which has been demonstrated to play a key role on the IL-1 production induced by LPS stimulation (10Han J. Lee J.-D. Bibbs L. Ulevitch R. Science. 1994; 265: 808-811Crossref PubMed Scopus (2446) Google Scholar, 11Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fischer J.E. Livi G.P. White J.R. Adam J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3181) Google Scholar). Anti-SAPK2/p38 antibodies were used to isolate this type of MAPK from lysates of cells exposed or not to colchicine. The immunoprecipitates were then incubated with [γ-32P]ATP and MBP, a standard substrate for MAPKs, to assess their phosphotransferase activity. Under these conditions, we failed to detect any increase of SAPK2 activity in the presence or in the absence of colchicine (Fig. 3 A). It is noticeable that the high basal activity was not inhibited by the addition of SB 203580, a specific inhibitor of SAPK2a and SAPK2b activities (11Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fischer J.E. Livi G.P. White J.R. Adam J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3181) Google Scholar), highly suggesting that these two activities were not activated upon microtubule disruption. This was confirmed by the lack of effect of SB 203580 on colchicine-induced IL-1β production, as evidenced in Fig. 3 B. We then envisaged that other congeners of the MAPK family could participate to the colchicine induction of IL-1 synthesis. To this end, anti-ERK1 and anti-ERK2 antibodies were used to immunoprecipitate the serine/threonine kinases from untreated or colchicine-treated cell lysates. The immunopellets were then incubated with [γ-32P]ATP and MBP. The data presented in Fig. 4 demonstrate that the treatment of THP1 cells with colchicine increases ERK1 (Fig. 4, A(upper) and B) and ERK2 (Fig. 4, A(lower) and B) kinase activities. Indeed, microtubule depolymerization promoted a swift activation of ERK2, which culminated at 2 min and then declined to the basal level by 60 min. In the case of ERK1, kinase activity was maximal by 5 min after colchicine treatment and persisted for 60 min, at variance with ERK2. We verified that lumicolchicine failed to activate any ERK activities (data not shown). MAPKs have been shown to phosphorylate and thereby to activate many regulatory proteins located in diverse cellular compartments, including nuclear transcriptional factors (20Seth A. Gonzalez F.A. Gupta S. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 24796-24804Abstract Full Text PDF PubMed Google Scholar, 21Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (858) Google Scholar). The nuclear content of phosphorylated MAPKs from cells treated or not with colchicine were assessed by Western blot analysis (Fig. 4 C, upper box), using antibodies recognizing the phosphorylated forms of ERK1 or ERK2. Following microtubule disruption, ERK1 and ERK2 translocated to the nucleus 10 min (lane 2) after colchicine treatment, according to their activation profile (Fig. 4, A and B). Augmentation of activated ERK in the nuclear compartment did not result from contaminating cytosolic proteins since the tubulin level, or Sos (data not shown) used as reporter proteins, remained constant in all conditions (Fig. 4 C, lower box). Activation of ERK1 and ERK2 seems to be mandatory for mediating the colchicine effect since PD 98059, a blocker of MEK-1 and MEK-2 (22Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3126) Google Scholar), abolished the stimulating effect that colchicine elicits on IL-1β production (Fig. 5, upper) and on IL-1β mRNA transcription (Fig. 5, lower) with an IC50 of 1 μm. To understand the mechanisms by which microtubule depolymerization stimulated ERKs, we studied the colchicine effect on the upstream acting MEK and Raf-1 kinase activities. It is well established that ERK1 and ERK2 are activated by the upstream MEK-1 and MEK-2 kinases (23Dérijard B. Raingeaud J. Barrett T. Wu I.-H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1431) Google Scholar, 24Winston B.W. Remigio L.D. Riches D.W.H. J. Biol. Chem. 1995; 270: 27391-27394Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The MEK-1 activity was measured in our system by mixing an anti-MEK-1 immunoprecipitate from colchicine-treated THP1 cell lysates with an immunopurified ERK1 inactive fraction obtained from unstimulated cell lysates. The data presented in Fig. 6 A(upper panel) provide evidence that microtubule depolymerization (lanes 2–6) stimulates MEK-1 activity. Phosphorylation of ERK1 used as substrate rose steeply after 5 min of treatment, to culminate at 30 min, and then slowly declined by 60 min. It is noteworthy that the time course of ERK1 phosphorylation in response to colchicine treatment paralleled that of MEK-1 autophosphorylation (Fig. 6, lower panel). Since Raf-1 activation sits upstream of the MEK activities, we sought to determine the colchicine effect on this activity. Therefore, Raf-1 was isolated from colchicine-treated THP1 cell lysates by using specific antibodies. Raf-1 kinase activity was then assessed by following phosphorylation of the unphosphorylated substrate MEK-1, obtained by previous immunoprecipitation from unstimulated THP1 cell lysates. A 6-fold increase in the extent of MEK phosphorylation was observed within the 5 min following addition of Raf-1 immunoprecipitated from colchicine-treated cell lysates to purified unstimulated MEK. This activation lasted for at least 30 min (Fig. 6 B). The mechanism of Raf-1 activation has been studied extensively over the past few years, and it is now clear that p21ras plays a key role in Raf-1 activation (25Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. Science. 1993; 260: 1658-1661Crossref PubMed Scopus (826) Google Scholar). Raf-1 has been shown to be recruited to the plasma membrane by p21ras when the latter binds to GTP (26Stokoe D. MacDonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (869) Google Scholar,27Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (909) Google Scholar). Thus, we determined whether colchicine was capable of modifying the relative level of p21ras bound to GTP. To this end, THP1 cells were labeled in vivo with [32P]orthophosphate, then subjected to colchicine treatment and lysed. Thereafter, p21ras was immunoprecipitated and the bound nucleotides were eluted and separated by thin layer chromatography. Under these conditions, we observed (Fig. 6 C) in unstimulated cells (lane 1) that p21ras was mainly associated to GDP (97%) and under microtubule depolymerization the proportion of p21ras-GTP complex increased in a discrete but reproducible (n = 4) manner from 3% to 5%. This extent of stimulation was similar to that observed after activation of insulin (28Burdering B.M.T. Medema R.H. Maassen J.A. Van de Wetering M.L. Van der Eb A.J. Bos J.L. EMBO J. 1991; 10: 1103-1109Crossref PubMed Scopus (226) Google Scholar), platelet-derived growth factor (29Satoh T. Endo M. Nakafuku M. Nakamura S. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5993-5997Crossref PubMed Scopus (200) Google Scholar), or epidermal growth factor (30Satoh T. Endo M. Nakafulu M. Akiyama T. Yamamoto T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7926-7929Crossref PubMed Scopus (210) Google Scholar) receptors. The accumulation of p21ras associated to GTP was maximal after 2 min of colchicine treatment and then declined to the basal level by 5 min. As noted above (Fig. 2), the stimulation of human monocytic cells by colchicine resulted in an increased tyrosine phosphorylation of a protein set, including proteins in the 55–60-kDa range, which are reminiscent of Src kinase congeners (31Rudd C.E. Janssen O. Prasad K.V.S. Raab M. Da Silva A. Telfer J.C. Yamamoto M. Biochim. Biophys. Acta. 1993; 1155: 239-266PubMed Google Scholar, 32Resh M.D. Cell. 1994; 76: 411-413Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 33Superti-Furga G. Courtneidge S.A. Bioessays. 1995; 17: 321-330Crossref PubMed Scopus (178) Google Scholar). We thus examined the ability of colchicine to stimulate the kinase activity of four members of the Src family that are expressed in THP1 cel

We have reported recently that colchicine and other microtubule-disrupting agents stimulated interleukin-1 (IL-1) alpha and beta synthesis in human monocytes. In this study we found that unexpectedly colchicine failed to stimulate the expression of two other potent immune mediators, namely tumor necrosis factor-alpha and interleukin-6. Remarkably, taxol which induces stable microtubule bundles, antagonized the colchicine but not the LPS-induced IL-1 synthesis. These results suggested that the colchicine-mediated IL-1 induction was generated by microtubule disassembly. We next demonstrated that microtubule disruption triggered an elevation of intracellular levels of cAMP and a subsequent stimulation of protein kinase A. The use of different protein kinase inhibitors supported a role of the PKA, but not the PKC, in the colchicine-induced IL-1 production. Furthermore, elevation of intracellular cAMP levels by 8-bromo-cAMP, forskolin, or cholera toxin potentiated the effect of suboptimal concentration of colchicine on IL-1 synthesis. However, these agents alone were unable to induce IL-1 synthesis. Therefore, our data indicate that the cAMP/protein kinase A-signaling pathway is necessary but not sufficient to generate IL-1 synthesis by microtubule disruption. Thus, microtubule-disrupting drugs appear as useful tools to further characterize the molecular events which regulate IL-1 production in human monocytes.

<h2>Summary</h2> Epigenetic deregulation of gene transcription is central to cancer cell plasticity and malignant progression but remains poorly understood. We found that the uncharacterized epigenetic factor chromodomain on Y-like 2 (CDYL2) is commonly over-expressed in breast cancer, and that high CDYL2 levels correlate with poor prognosis. Supporting a functional role for CDYL2 in malignancy, it positively regulated breast cancer cell migration, invasion, stem-like phenotypes, and epithelial-to-mesenchymal transition. CDYL2 regulation of these plasticity-associated processes depended on signaling via p65/NF-κB and STAT3. This, in turn, was downstream of CDYL2 regulation of <i>MIR124</i> gene transcription. CDYL2 co-immunoprecipitated with G9a/EHMT2 and GLP/EHMT1 and regulated the chromatin enrichment of G9a and EZH2 at <i>MIR124</i> genes. We propose that CDYL2 contributes to poor prognosis in breast cancer by recruiting G9a and EZH2 to epigenetically repress <i>MIR124</i> genes, thereby promoting NF-κB and STAT3 signaling, as well as downstream cancer cell plasticity and malignant progression.

We have characterized a T lymphocyte endopeptidase activity that hydrolyses succinyl-alanine-alanine-phenylalanine-paranitroanilide (Suc-Ala-Ala-Phe-pNa). Hydrolysis of this substrate by intact Jurkat T cells was markedly enhanced when exogenous aminopeptidase N was added to the incubation medium. It thus appears that the release of paranitroaniline from Suc-Ala-Ala-Phe-pNA results from the combination of two distinct enzymatic activities: (i) an endopeptidase activity that cleaves the substrate at the alanyl bond and (ii) an aminopeptidase activity that ultimately cleaves the phenylalanyl bond. This cleavage was further confirmed by HPLC analysis. Specific endopeptidase 24.11 inhibitors were shown to inhibit the endopeptidase activity. These features are reminiscent of the characteristics of neutral endopeptidase (NEP, also known as endopeptidase 24.11, CALLA or CD10). Anti-CD10 monoclonal antibodies (mAbs) recognized the CD10+ B cell line Raji, but not Jurkat cells as assessed by FACS analysis. This is probably due to a lack of sensitivity of this method, the level of NEP activity in Jurkat T cells being 3-5% of that measured in B cell lines. Anti-CD10 mAbs immunoprecipitated endopeptidase 24.11 activities in both Jurkat T cells and Raji B cells, demonstrating that T lymphocytes express a CALLA-related endopeptidase. We also demonstrate that T and B cell endopeptidases have the same molecular weight, that T cells express less functional CALLA mRNA than B cells and that there are at least two shorter transcripts (1.8 and 0.8 kb) in both T and B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Nutrient availability is a key determinant of tumor cell behavior. While nutrient‐rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor general control non‐derepressible 2 (GCN2; also known as eIF‐2‐alpha kinase 4) represses the expression of the precursor of ribosomal RNA (rRNA), 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53‐mediated apoptosis in patient‐derived models of colon adenocarcinoma (COAD). In nutrient‐rich conditions, a cell‐autonomous GCN2 activity supports cell proliferation by stimulating 47S rRNA transcription, independently of the canonical integrated stress response (ISR) axis. Impairment of GCN2 activity prevents nuclear translocation of methionyl‐tRNA synthetase (MetRS), resulting in nucleolar stress, mTORC1 inhibition and, ultimately, autophagy induction. Inhibition of the GCN2–MetRS axis drastically improves the cytotoxicity of RNA polymerase I (RNA pol I) inhibitors, including the first‐line chemotherapy oxaliplatin, on patient‐derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls ribosome biogenesis according to the nutritional context. Furthermore, pharmacological co‐inhibition of the two GCN2 branches and RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically stressed COAD cells.

Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation. Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation. The RET 4The abbreviations used are:RETrearranged during transfectionDRMsdetergent-resistant membranesERKextracellular signal regulated kinaseGDNFglial cell line-derived neurotrophic factorGFRαglial cell line-derived neurotrophic factor receptor αMBCmethyl-β-cyclodextrineMEN2multiple endocrine neoplasia type 2PI3Kphosphatidylinositol 3 kinasePLCγphospholipase C γShcSrc homology and collagen proteinTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingGFLGDNF family ligandsHAhemagglutininMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideAPAP20187. 4The abbreviations used are:RETrearranged during transfectionDRMsdetergent-resistant membranesERKextracellular signal regulated kinaseGDNFglial cell line-derived neurotrophic factorGFRαglial cell line-derived neurotrophic factor receptor αMBCmethyl-β-cyclodextrineMEN2multiple endocrine neoplasia type 2PI3Kphosphatidylinositol 3 kinasePLCγphospholipase C γShcSrc homology and collagen proteinTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingGFLGDNF family ligandsHAhemagglutininMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideAPAP20187. proto-oncogene is located on chromosome 10q11.2 and encodes a receptor tyrosine kinase with four cadherin-related motifs and a cysteine-rich domain in the extracellular domain (1Airaksinen M.S. Saarma M. Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1445) Google Scholar). It associates with ligand-specific co-receptors known as GFRαs (GDNF, glial-cell-line-derived neurotrophic factor, family receptors α), to form functional receptors for the GFL (GDNF family ligands). In the current view, homodimeric GFL binding induces a GFL2-GFRα2-RET2 complex (2Leppanen V.M. Bespalov M.M. Runeberg-Roos P. Puurand U. Merits A. Saarma M. Goldman A. EMBO J. 2004; 23: 1452-1462Crossref PubMed Scopus (52) Google Scholar). RET dimerization leads to increased receptor kinase activity and autophosphorylation of cytoplasmic tyrosine residues, which serve as docking sites for Src homology 2 (SH2)- and phospho-tyrosine-binding domain-containing proteins, such as Shc or phospholipase Cγ (3Ichihara M. Murakumo Y. Takahashi M. Cancer Lett. 2004; 204: 197-211Crossref PubMed Scopus (162) Google Scholar). These proteins then recruit additional effector molecules, resulting in the assembly of signaling complexes and the activation of intracellular signaling pathways, including the Ras/extracellular-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. GFL-mediated signaling pathways are involved in the development and maintenance of a broad spectrum of neuronal subpopulations (1Airaksinen M.S. Saarma M. Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1445) Google Scholar). rearranged during transfection detergent-resistant membranes extracellular signal regulated kinase glial cell line-derived neurotrophic factor glial cell line-derived neurotrophic factor receptor α methyl-β-cyclodextrine multiple endocrine neoplasia type 2 phosphatidylinositol 3 kinase phospholipase C γ Src homology and collagen protein terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling GDNF family ligands hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide AP20187. rearranged during transfection detergent-resistant membranes extracellular signal regulated kinase glial cell line-derived neurotrophic factor glial cell line-derived neurotrophic factor receptor α methyl-β-cyclodextrine multiple endocrine neoplasia type 2 phosphatidylinositol 3 kinase phospholipase C γ Src homology and collagen protein terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling GDNF family ligands hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide AP20187. Recently, membrane microdomains, or lipid rafts, have been shown to profoundly influence the functional impact of GDNF-stimulated RET downstream signaling (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Lipid rafts are suggested to be lateral microdomains in membranes of living cells, enriched in sphingolipids, cholesterol, and specific membrane proteins. They are characterized by higher order and by having a lower buoyant density than bulk plasma membranes (6Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5149) Google Scholar, 7Sprong H. van der Sluijs P. van Meer G. Nat. Rev. Mol. Cell. Biol. 2001; 2: 504-513Crossref PubMed Scopus (476) Google Scholar). Although uncertainties about the precise molecular nature of rafts remain (8Edidin M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 257-283Crossref PubMed Scopus (1134) Google Scholar, 9Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 10Sharma P. Varma R. Sarasij R.C. Ira Gousset K. Krishnamoorthy G. Rao M. Mayor S. Cell. 2004; 116: 577-589Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 11Kusumi A. Koyama-Honda I. Suzuki K. Traffic. 2004; 5: 213-230Crossref PubMed Scopus (333) Google Scholar), compelling evidence indicates that lipid rafts can coalesce into larger and more stable structures where proteins can segregate to perform functions (12Golub T. Wacha S. Caroni P. Curr. Opin. Neurobiol. 2004; 14: 542-550Crossref PubMed Scopus (135) Google Scholar, 13Rajendran L. Simons K. J. Cell Sci. 2005; 118: 1099-1102Crossref PubMed Scopus (468) Google Scholar). With regard to GDNF-stimulated RET signaling, it has been shown that at steady state, GFRα1, but not RET, is a resident of lipid raft-related detergent-resistant membranes (DRMs). GDNF stimulation was demonstrated to trigger RET recruitment and activation into DRMs. This was strongly correlated with downstream signaling, cell survival, and differentiation (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Furthermore, Paratcha et al. (5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) showed that RET association with DRMs may influence the nature of the intracellular signaling. Together, these studies demonstrate a connection between RET association with DRMs and RET signaling and support a role for lipid rafts in controlling GDNF-stimulated RET signaling. Mutations in the RET proto-oncogene have been identified as causative for human papillary thyroid carcinoma, multiple endocrine neoplasia (MEN) type 2A and 2B, and familial medullary thyroid carcinoma (14Hansford J.R. Mulligan L.M. J. Med. Genet. 2000; 37: 817-827Crossref PubMed Scopus (208) Google Scholar). MEN2A is an autosomal dominant cancer syndrome, characterized by medullary thyroid carcinoma, pheochromocytoma, and hyperplasia of the parathyroid gland. MEN2A mutations were identified in the cysteine-rich region, and ∼90% of those mutations affect codon 634 (most frequently a cysteine to arginine change) (15Eng C. J. Clin. Oncol. 1999; 17: 380-393Crossref PubMed Google Scholar). They result in a constitutive activation of RET through formation of covalently linked dimers of the receptor, independent of GFL (16Asai N. Iwashita T. Matsuyama M. Takahashi M. Mol. Cell. Biol. 1995; 15: 1613-1619Crossref PubMed Google Scholar, 17Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Crossref PubMed Scopus (796) Google Scholar). Dimerization occurs early during the maturation process and results in additional activation of incompletely glycosylated intracellular RET precursors (18Cosma M.P. Cardone M. Carlomagno F. Colantuoni V. Mol. Cell. Biol. 1998; 18: 3321-3329Crossref PubMed Scopus (51) Google Scholar). Whether MEN2A signaling is identical to GFL-triggered signaling is difficult to answer because activation of the constitutive MEN2A protein cannot be triggered in resting cells, thus precluding a qualitative and quantitative comparison with the GFL-inducible signaling pathways. Consequently, the current model to explain how MEN2A RET mutants promote tumorigenesis only considers the permanent nature of MEN2A signaling. To address this issue, we have generated a chimeric RET-Fv protein that can be alternatively activated in the same cell, either with the natural ligand GDNF or with a synthetic bivalent dimerizing ligand (19Clackson T. Yang W. Rozamus L.W. Hatada M. Amara J.F. Rollins C.T. Stevenson L.F. Magari S.R. Wood S.A. Courage N.L. Lu X. Cerasoli Jr., F. Gilman M. Holt D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10437-10442Crossref PubMed Scopus (418) Google Scholar). The latter mimicked ligand-independent RET dimerization by MEN2A mutants. This induced activation of RET specifically resulted in altered AKT activation but not ERK activation. Furthermore, we demonstrated that a lipid-based cholesterol-sensitive environment regulates the tuning of GDNF-induced AKT activation, suggesting a role of lipid rafts. Finally, we showed that MEN2A mutants escape from this control step. Therefore, these studies revealed complex alterations in oncogenic signaling by MEN2A RET mutants. Antibodies, Reagents, and DNA Constructs—Phospho-tyrosine (4G10), hemagglutinin (HA), flotillin, and human transferrin receptor antibodies were from Upstate Biotechnology (Lake Placid, NY), Covance Research (Berkeley, CA), BD Transduction Laboratories, and Zymed Laboratories (South San Francisco, CA), respectively. Protein-specific anti-phospho antibodies were from Cell Signaling Technology (Beverly, MA). Anti-RET was described elsewhere (20Pasini A. Geneste O. Legrand P. Schlumberger M. Rossel M. Fournier L. Rudkin B.B. Schuffenecker I. Lenoir G.M. Billaud M. Oncogene. 1997; 15: 393-402Crossref PubMed Scopus (87) Google Scholar). Anti-phospho-RETs (Tyr-1015 or Tyr-1062) were generously provided by Dr. Massimo Santoro (21Salvatore D. Barone M.V. Salvatore G. Melillo R.M. Chiappetta G. Mineo A. Fenzi G. Vecchio G. Fusco A. Santoro M. J. Clin. Endocrinol. Metab. 2000; 85: 3898-3907Crossref PubMed Scopus (56) Google Scholar). Lipofectamine 2000, human GDNF, protease inhibitors, monensin, and brefeldin A were from Invitrogen, Promega Corp. (Madison, WI), Roche Diagnostics, BD Biosciences, and Calbiochem, respectively. MTT, Triton X-100, methyl-β-cyclodextrin (MBC), and anisomycin were from Sigma. AP20187 (AP) was provided by Ariad Pharmaceuticals (Cambridge, MA). The HA-tagged AP-binding domain (Fv) was subcloned from the Ariad pC4-Fv1E vector, into the ApaI/XbaI sites of the pcDNA3.1(+) vector (Invitrogen). The Ret9 cDNA (20Pasini A. Geneste O. Legrand P. Schlumberger M. Rossel M. Fournier L. Rudkin B.B. Schuffenecker I. Lenoir G.M. Billaud M. Oncogene. 1997; 15: 393-402Crossref PubMed Scopus (87) Google Scholar) was mutated by PCR to suppress the stop codon and subcloned into the HindIII/XbaI sites of the pcDNA-Fv vector. The HA-GFRα1 cDNA kindly provided by Dr. Carlos Ibanez (22Trupp M. Raynoschek C. Belluardo N. Ibanez C.F. Mol. Cell. Neurosci. 1998; 11: 47-63Crossref PubMed Scopus (162) Google Scholar) was subcloned into the BamHI/EcoRI sites of the pBabe retroviral vector (23Morgenstern J.P. Land H. Nucleic. Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1898) Google Scholar). Cell Culture and Transfections—Rat-1 cells, immortalized rat fibroblasts expressing neither Ret nor GFRα1, were maintained in complete medium: Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics (all from Sigma). Cells were transfected with Ret-Fv- and GFRα1-containing vectors with the use of Lipofectamine according to manufacturer's recommendations. Stably transfected cells were selected in complete medium with G418 (1 mg/ml) and/or puromycin (2 μg/ml) (both from Sigma). Rat-1 cell clones transformed by RET MEN2A mutants (24Segouffin-Cariou C. Billaud M. J. Biol. Chem. 2000; 275: 3568-3576Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) were maintained in complete medium with puromycin (2 μg/ml). MEN2A cells were further transfected with GFRα1, and stable clones (termed Mα) were selected with G418 (1 mg/ml) and puromycin (2 μg/ml). Stimulations and Immunoblotting—Equal numbers of cells were seeded in plates to achieve ∼60% confluence in 24 h and serum-starved for 4 h prior to stimulation. Cells were stimulated for the indicated period of time with either 10 ng/ml GDNF or 100 nm AP20187 in serum-free medium and lysed at 4 °C for 20 min in radioimmune precipitation buffer (150 mm NaCl, 50 mm Tris-HCl (pH 7.2), 1% Triton X-100, 1% Sodium deoxycholate, 0.05% SDS, 4 mm NaVO4, 5 mm EGTA, and protease inhibitors). Precleared lysates (10 min at 13,000 rpm) were subjected to SDS-PAGE and Western blotting using the relevant antibody as described previously (25Vidalain P.O. Azocar O. Servet-Delprat C. Rabourdin-Combe C. Gerlier D. Manie S. EMBO J. 2000; 19: 3304-3313Crossref PubMed Scopus (165) Google Scholar). Densitometry of the blots was assessed using a Fluor-S multi-imager system (Bio-Rad). DRM Assay—Cells were scrapped, rinsed once with phosphate-buffered saline, and lysed at 3 mg of protein/ml in a final volume of 0.5 ml of ice-cold TNE buffer (150 mm NaCl, 25 mm Tris-HCl (pH 7.2), 4 mm NaVO4, 5 mm EGTA, and protease inhibitors) containing 0.5% Triton X-100. Lysates were homogenized five times through a 23-gauge needle and incubated with constant agitation for 30 min at 4 °C. They were then mixed with 1 ml of 60% saccharose in TNE buffer containing 0.1% Triton X-100 and transferred to ultracentrifuge tubes. The samples were overlaid by a saccharose gradient (2 ml of 30% saccharose followed by 1 ml of 25, 20, 17.5, 15, 12.5, and 5% saccharose in TNE buffer containing 0.1% Triton X-100) and centrifuged for 16 h at 39,000 rpm in a Beckman SW41 rotor at 4 °C. The gradient was fractionated into 1-ml fractions (see Fig. 6B, 1.15-ml fractions) from the top of the tube, except for the last one that contained 1.5 ml. Aliquots of each fraction were analyzed by SDS-PAGE and immunoblotting. Cellular Cholesterol Depletion and Brefeldin A/Monensin Treatment—Cells prepared as described above were incubated in serum-free medium containing MBC (10 mm) and 50 mm Hepes for 20 min at 37 °C with constant agitation. Cells were then rinsed twice in phosphate-buffered saline, stimulated, and lysed to isolate DRMs. To abrogate RET protein maturation, cells were treated or not with 2.5 μg/ml brefeldin A and 0.66 μl/ml monensin (Golgi stop) in serum-free Dulbecco's modified Eagle's medium for 6 h and further stimulated with AP for the indicated period of time, before being processed for immunoblotting analysis. Cell Growth and TUNEL Assays—Cells were plated at 500 cells/well in 96-well plates in complete medium with GDNF (10 ng/ml) or AP (100 nm), the growth medium being changed every 3 days. Every 2 days, cells were harvested, and an MTT test was realized for each condition of stimulation; 30 μl of MTT (7.5 mg/ml in phosphate-buffered saline) was added to each well, and cells were incubated for 4 h at 37°C. The medium was then replaced with 100 μl of Me2SO with 0.04 n HCl. OD was read at 492 nm. The measured absorbance is proportional to the number of live cells present. For TUNEL assay, Mα cells were plated on coverslips at 7 × 104 cells/well in Dulbecco's modified Eagle's medium containing 1% fetal bovine serum, overnight, and stimulated with 50 ng/ml GDNF for 1 h in the same medium before being incubated with anisomycin (1 μg/ml) for 5 h. A TUNEL assay was performed according to the supplier's instructions (in situ cell death detection kit, Roche Diagnostics). Controlled Homodimerization and Activation of RET—The inducible GDNF/GFRα1-independent activation system of RET is controlled by the synthetic bivalent dimerizing ligand AP. It is based on the ability of a fusion protein (RET-Fv), containing RET linked to an AP-binding domain (Fv), to be induced to homodimerize in the presence of AP, leading to RET activation (Fig. 1A). Both RET-Fv and GFRα1 were stably expressed in Rat-1 fibroblastic cells so that RET-Fv can be activated alternatively with GDNF or AP treatment. Fig. 1A shows the expression of RET-Fv and GFRα1 in representative Rat-1 fibroblast clones. The two RET species correspond to the incompletely glycosylated protein present in the endoplasmic reticulum (162 kDa, major form in Rat-1 cells) and to the fully glycosylated protein expressed at the plasma membrane (182 kDa) (26van Weering D.H. Moen T.C. Braakman I. Baas P.D. Bos J.L. J. Biol. Chem. 1998; 273: 12077-12081Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Similarly, the 65-kDa form of GFRα1 was expressed on the cellular surface, whereas the 55-kDa form was not (not shown) and was assumed to correspond to an immature GFRα1 product. Stimulation of RET-Fv/GFRα1-expressing cells with an optimal concentration of AP or GDNF for 15 min induced a comparable activation of mature 182-kDa RET proteins, as detected with an antibody specific to phosphorylated autocatalytic residue Y905RET (Fig. 1B, upper panel). However, in contrast to GDNF, AP diffused into the cytoplasm and then bound to and activated the immature 162-kDa Ret protein. This is similar to what is typically observed with MEN2A RET mutants in which both the mature and the immature forms of RET are activated. Despite the additional activation of the immature form of RET, a comparable increase in tyrosine phosphorylation of cellular proteins was detected after either GDNF or AP stimulation (Fig. 1B, lower panel). It would appear that the addition of the Fv module to the cytoplasmic tail of RET does not obviously alter RET activation and signaling since a similar pattern of tyrosine phosphorylation was observed when cells were transfected with wild type RET instead of RET-Fv and stimulated with GDNF (Fig. 1B). Taken together, these results indicated that homodimerization of RET can lead to its efficient activation without the requirement for GDNF/GFRα1 complex. They also suggested that activation of the intracellular immature 162-kDa Ret protein does not contribute much to the RET-dependent pattern of tyrosine phosphorylation. GDNF/GFRα1-independent RET Activation Leads to an Altered Cellular Outcome—Next, we investigated whether activation of RET by AP or GDNF can differently affect biological outcomes. RET-Fv/GFRα1-expressing cells were chronically stimulated with either GDNF or AP, and the growth rate was monitored. Neither AP nor GDNF stimulated growth rate during the first 6 days of culture (Fig. 2). After 8 days of culture, both non-stimulated and GDNF-stimulated cells grew in an even monolayer and displayed normal contact inhibition, as shown on representative May-Grunwald-Giemsa staining of cells after 10 days of culture. As a consequence, these cells stopped growing. In contrast, AP-stimulated cells began to overgrow one another, indicating that they had lost the ability to be contact inhibited. These results indicated the existence of specific AP-mediated cellular outcome(s), suggesting that GDNF/GFRα1-independent RET activation leads to alterations of intracellular signaling pathways. GDNF/GFRα1-independent RET Activation Impacts on AKT Regulation—We next tested whether activation of RET by AP or GDNF can translate into different, or differently regulated, RET-mediated signaling pathways. We first monitored the extent of RET autophosphorylation using two other antibodies specific to phosphorylated Y1015RET and Y1062RET. Fig. 3A shows that phosphorylation over a time course of these autocatalytic residues was comparably achieved by GDNF or AP stimulation. Together with the comparable phosphorylation of Y905RET depicted in Fig. 1, these results indicate that AP or GDNF stimulation leads to similar activation of RET tyrosine kinase. It is of note that RET phosphorylation in Rat-1 cells remained high after 2 h of stimulation and that it could last for 6 h before declining (see Fig. 6A). Similar results were obtained with wild type RET instead of RET-Fv (not shown), indicating that the Fv-binding module is not involved in the long lasting phosphorylation of RET in Rat-1 cells. Y1015RET and Y1062RET are binding sites for PLCγ and Shc/Dok4–5/IRS-1/FRS-2, respectively. The bound molecular partners are in turn phosphorylated by RET (3Ichihara M. Murakumo Y. Takahashi M. Cancer Lett. 2004; 204: 197-211Crossref PubMed Scopus (162) Google Scholar). Fig. 3A shows that both Shc and PLCγ were phosphorylated in a comparable manner following GDNF or AP stimulation and that their kinetics of phosphorylation paralleled those of RET. These results implied that RET activation by AP or GDNF does not influence proximal RET signaling, i.e. autophosphorylation of RET and the resulting phosphorylation of the molecular partners Shc and PLCγ. We next examined the activation of downstream pathways by looking at ERK and AKT phosphorylation. GDNF induced a transient phosphorylation of ERK and AKT (Fig. 3). Therefore, both Ras/ERK and PI3K/AKT signaling pathways can be attenuated even with the prolonged phosphorylation of RET observed in Rat-1 cells. As compared with GDNF, AP stimulated ERK phosphorylation to a similar amplitude and duration. However, in marked contrast, the kinetics of AP-stimulated AKT phosphorylation was delayed and sustained for several hours (Fig. 3, A and B). Kinetic alteration of AKT phosphorylation was detected on both serine 473 and threonine 308, which additively activate AKT (28Brazil D.P. Yang Z.Z. Hemmings B.A. Trends Biochem. Sci. 2004; 29: 233-242Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar). It should be emphasized that AP does not directly affect AKT activation since no alteration of AKT phosphorylation could be detected following GDNF treatment of cells expressing RET (instead of RET-Fv) in the presence of AP (not shown). These results indicated that GDNF/GFRα1-independent RET activation translates into differently regulated AKT activation, without really affecting ERK activation. Neither Signaling from GFRα1 Alone nor from Immature Forms of RET Contributes to AKT Phosphorylation—Since GDNF has been reported to signal independently of RET via GFRα1 (29Trupp M. Scott R. Whittemore S.R. Ibanez C.F. J. Biol. Chem. 1999; 274: 20885-20894Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 30Poteryaev D. Titievsky A. Sun Y.F. Thomas-Crusells J. Lindahl M. Billaud M. Arumae U. Saarma M. FEBS Lett. 1999; 463: 63-66Crossref PubMed Scopus (143) Google Scholar), it was important to evaluate whether signaling originating from GFRα1 alone could contribute to AKT and ERK phosphorylation in Rat-1 cells. We thus made use of Rat-1 clones expressing GFRα1 only and have been unable to detect any phosphorylation of AKT and ERK following GDNF stimulation of these clones (Fig. 4A). Next, we evaluated whether AP-activated immature 162-kDa forms of RET-Fv were involved in the differential phosphorylation of AKT. To address this issue, cells were treated with brefeldin A and monensin to abrogate RET maturation and delivery to the plasma membrane (31Jung T. Schauer U. Heusser C. Neumann C. Rieger C. J. Immunol. Methods. 1993; 159: 197-207Crossref PubMed Scopus (903) Google Scholar). The combined use of the two protein maturation blocking drugs was favored because under our experimental conditions, each drug alone was not efficient enough to fully prevent apparition of mature RET proteins (not shown). Cellular treatment for 6 h with the two drugs efficiently blocked the expression of the plasma membrane-localized mature forms of RET while increasing the expression of the endoplasmic reticulum-associated immature 162-kDa form of RET (Fig. 4B). Under these conditions, AP stimulation of cells could still induce phosphorylation of the immature form. Interestingly, phosphorylation of Shc was also detectable, indicating that activated endoplasmic reticulum-associated immature 162-kDa forms of RET can recruit molecular partners. However, these activated forms of RET made no detectable contribution to AKT and ERK activation. These data, together with the observation that additional activation of immature forms of RET did not modify the tyrosine phosphorylation pattern (Fig. 1B), strongly suggest that immature forms of RET do not contribute to the signaling pathways monitored here. GDNF/GFRα1-independent RET Activation Does Not Associate with DRMs—GDNF/GFRα1-stimulated RET associates with the raft-related DRMs, and this association has been correlated with the control of downstream signaling (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Thus, to gain an insight into the mechanism(s) responsible for the differential regulation of AKT activation, we asked whether AP stimulation could also induce the association of RET with DRMs. As expected, GDNF stimulation induced an association of mature forms of RET-Fv with DRMs (Fig. 5A). Flotillin and transferrin receptor were used as positive and negative markers, respectively, of DRM-associated proteins. Although RET must associate with GFRα1to be activated by GDNF, more than 60% of phosphorylated RET-Fv was recovered in soluble fractions (Fig. 5B). This does not seem to reflect a large movement of RET out of DRMs after recruitment and activation since RET remained associated with DRMs for several hours after the beginning of the stimulation in Rat-1 cells (Fig. 5B, and see also Fig. 6A). These results suggested that RET association with DRMs is weaker than GFRα1 association and that it can be partly disrupted by non-ionic detergents. In marked contrast to these results, RET association with DRMs was not evident upon AP stimulation. Similarly, RET proteins carrying a representative MEN2A mutation at Cys-634 (24Segouffin-Cariou C. Billaud M. J. Biol. Chem. 2000; 275: 3568-3576Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) were not recovered in the DRM fractions (Fig. 5A). These results clearly demonstrated a difference in the association (or the strength of association) of RET with DRMs between GDNF-dependent and GDNF/GFRα1-independent RET activation. RET Association with DRMs Correlates with Specific Regulation of AKT—The above results raised the possibility that tight regulation of AKT, but not ERK, phosphorylation is linked to DRM association of RET. This hypothesis was tested in two different ways. First, association of proteins with DRMs has been shown to be sensitive to the cholesterol-depleting drug MBC (32Xavier R. Brennan T. Li Q. McCormack C. Seed B. Immunity. 1998; 8: 723-732Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). Preincubation of Rat-1 cells for 20 min with 10 mm MBC before stimulation did not affect RET phosphorylation and its

Abstract The metalloprotease-dependent extracellular domain cleavage of the adhesion molecule CD44 is frequently observed in human tumors and is thought to promote metastasis. This cleavage is followed by γ-secretase-dependent release of CD44 intracellular domain (CD44-ICD), which exhibits nuclear signaling activity. Using a reversible Ret-dependent oncogenic conversion model and a restricted proteomic approach, we identified a positive correlation between the neoplastic transformation of Rat-1 cells and the expression of standard CD44. In these transformed cells, CD44 was found to undergo a sequential metalloprotease and γ-secretase cleavage, resulting in an increase in expression of CD44-ICD. We showed that this proteolytic fragment possesses a transforming activity. In support of this role, a significant and specific reduction in Ret-induced transformation of Rat-1 cells was observed following drug-mediated inhibition of γ-secretase. Taken together, these findings suggest that the shedding of CD44 may not only modulate metastasis but also affects earlier events in tumorigenesis through the release of CD44-ICD. (Cancer Res 2006; 66(7): 3681-7)

MyD88 is an adaptor molecule in Toll-like receptor and interleukin 1 receptor signaling implicated in tumorigenesis through proinflammatory mechanisms. We have recently reported that MyD88 also directly promotes optimal activation of the Ras/Erk pathway. Here we investigate MyD88 implication in the maintenance of the transformation of Ras-dependent tumors.RNA interference was used to inhibit MyD88 expression in the colon cancer cell lines HCT116 and LS513. Apoptosis, DNA damage, p53 function, ERCC1 levels, and Ras and inflammatory signaling pathways were analyzed. Using in vitro assays and xenotransplantation in nude mice (five per group), HCT116 tumor growth was assessed following MyD88 knockdown in presence or absence of chemotherapy.MyD88 exerts antiapoptotic functions in colon cancer cells via the Ras/Erk, but not the NF-κB, pathway. MyD88 inhibition leads to defective ERCC1-dependent DNA repair and to accumulation of DNA damage, resulting in cancer cell death via p53. Furthermore, we show that knocking down MyD88 sensitizes cancer cells to genotoxic agents such as platinum salts in vitro and in vivo. Indeed, HCT116 tumor growth following treatment with a combination of suboptimal MyD88 inhibition and suboptimal doses of cisplatin (fold tumor increase = 5.4 ± 1.6) was statistically significantly reduced in comparison to treatment with doxycycline alone (12.4 ± 3.1) or with cisplatin alone (12.5 ± 2.6) (P = .005 for both, one-sided Student t test).Collectively, these results indicate a novel and original link between inflammation, DNA repair, and cancer, and provide further rationale for MyD88 as a potential therapeutic target in Ras-dependent cancers, in the context of concomitant genotoxic chemotherapy.

Engagement of β1 integrins in terminally differentiated human B cell lines, such as ARH-77, leads to prominent tyrosine phosphorylation of the p130 Crk-associated substrate (Cas). Cas regulates the assembly of several SH2 and SH3 domain-containing proteins into signaling complexes, which are potentially involved in the propagation of downstream signals. We demonstrate here that immunoprecipitated Cas from β1 integrin-stimulated ARH-77 cells was associated with tyrosine kinase and phosphatase activities and that integrin ligation led to the recruitment of at least p59Fyn tyrosine kinase and SHP2 tyrosine phosphatase in Cas immune complexes. Cotransfection studies in COS-7 cells further indicated that Fyn/Cas physical interaction and Fyn-mediated Cas phosphorylation required amino acids 638–889 in the C-terminal region of Cas. This sequence contains both c-Src SH2 and SH3 domain-binding motifs. In vitro binding studies using glutathioneS-transferase fusion proteins derived from the SH2 or SH3 domains of Fyn suggested that both Fyn domains can participate in Fyn/Cas interaction. These data implicate Fyn and SHP2 as potential modulators of Cas signaling complexes in B cells. Engagement of β1 integrins in terminally differentiated human B cell lines, such as ARH-77, leads to prominent tyrosine phosphorylation of the p130 Crk-associated substrate (Cas). Cas regulates the assembly of several SH2 and SH3 domain-containing proteins into signaling complexes, which are potentially involved in the propagation of downstream signals. We demonstrate here that immunoprecipitated Cas from β1 integrin-stimulated ARH-77 cells was associated with tyrosine kinase and phosphatase activities and that integrin ligation led to the recruitment of at least p59Fyn tyrosine kinase and SHP2 tyrosine phosphatase in Cas immune complexes. Cotransfection studies in COS-7 cells further indicated that Fyn/Cas physical interaction and Fyn-mediated Cas phosphorylation required amino acids 638–889 in the C-terminal region of Cas. This sequence contains both c-Src SH2 and SH3 domain-binding motifs. In vitro binding studies using glutathioneS-transferase fusion proteins derived from the SH2 or SH3 domains of Fyn suggested that both Fyn domains can participate in Fyn/Cas interaction. These data implicate Fyn and SHP2 as potential modulators of Cas signaling complexes in B cells. Integrins are α/β heterodimeric adhesion receptors that are involved in cell/cell and cell/matrix interactions (1Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5816) Google Scholar, 2Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar). With regard to B lymphocytes, integrins are involved in cell localization within specific microenvironments (3Freedman A.S. Munro M.J. Rice G.E. Bevilacqua M.P. 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Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar). In many cell types, including B lymphocytes, there is prominent tyrosine phosphorylation of proteins of 105–130 kDa. Several of these substrates have been identified, including p125FAK(FAK (focal adhesion kinase)) (10Schaller M. Borgman C. Cobb B. Vines R. Reynolds A. Parsons J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1280) Google Scholar, 11Hamawy M.M. Mergenhagen S.E. Siraganian R.P. J. Biol. Chem. 1993; 268: 6851-6854Abstract Full Text PDF PubMed Google Scholar, 12Huang M. Lipfert L. Cunningham M. Brugge J. Ginsberg M. Shattil S. J. Cell Biol. 1993; 122: 473-483Crossref PubMed Scopus (155) Google Scholar, 13Lipfert L. Haimovich B. Schaller M. Cobb B. Parsons J. Brugge J. J. Cell Biol. 1992; 119: 905-912Crossref PubMed Scopus (624) Google Scholar, 14Manié S. Astier A. Wang D. Phifer J. Chen J. Lazarovits A. Morimoto C. Freedman A. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar); RAFTK (related adhesionfocal tyrosine kinase; also known as PYK2 and CAKβ) (15Avraham S. London R. Fu Y. Ota S. Hiregowdara D. Li J. Jiang S. Pasztor L.M. White R.A. Groopman J.E. Avraham H. J. Biol. Chem. 1995; 270: 27742-27751Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 16Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J. Plowman G. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1243) Google Scholar, 17Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 18Astier A. Avraham H. Manié S.N Groopman J. Canty T. Avraham S. Freedman A.S. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar); p120c- cbl, the cellular homologue of the oncogene v-cbl (19Manié S. Sattler M. Astier A. Phifer J. Canty T. Morimoto C. Druker B. Salgia R. Griffin J. Freedman A. Exp. Hematol. 1997; 25: 45-50PubMed Google Scholar); p130Cas (Cas (Crk-associatedsubstrate)) (20Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 21Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 22Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 23Petruzzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar); and the Cas-like molecule p105HEF1 (human enhancer offilamentation 1), also known as Cas-L for lymphocyte-type Cas protein (24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 25Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (146) Google Scholar). Cas was originally identified as one of the major tyrosine-phosphorylated proteins in v-crk- or v-src-transformed cells (26Kanner S. Reynolds A. Wang H.-C.R. Vines R. Parsons J. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 27Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). Cas belongs to a new family of structurally related proteins that are thought to act as “docking molecules,” i.e. regulating the assembly of several SH2 and SH3 domain-containing proteins into signaling complexes. This family includes three members so far: Cas, HEF1/Cas-L (25Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (146) Google Scholar, 28Law S. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (219) Google Scholar), and Efs/Sin (embryonal Fyn-associatedsubstrate/Src-interacting orsignal-integrating protein) (29Ishino M. Ohba T. Sasaki H. Sasaki T. Oncogene. 1995; 11: 2331-2338PubMed Google Scholar, 30Alexandropoulos K. Baltimore D. Genes Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar). They all contain an SH3 domain in the N-terminal region; a cluster of SH2 domain-binding motifs that have been named the “substrate domain” (27Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar); and, with the exception of HEF1/Cas-L, several potential binding motifs for SH3 domains. Following β1 integrin cross-linking, Cas phosphorylation was most prominent in B cell lines representative of a more differentiated state, such as the multiple myeloma cell lines ARH-77, IM-9, and RPMI 8226, and was minimally detectable in normal mature B cells (24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In contrast, HEF1 was consistently tyrosine-phosphorylated in all immature, mature, and terminally differentiated B cell lines as well as in normal B cells following both β1 integrin and B cell antigen receptor ligation. Therefore, the phosphorylation of these two related molecules appears to be differentially regulated in B cells. Proteins interacting with Cas include FAK and PTP-1B, which bind to the Cas SH3 domain (31Polte T. Hanks S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (384) Google Scholar, 32Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 33Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar); Crk family members, which interact with tyrosine-phosphorylated Cas through SH2 domain-binding motifs (23Petruzzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,34Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar, 35Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 36Burnham M. Harte M. Richardson A. Parsons J. Bouton A. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar); c-Src, which interacts with tyrosine-phosphorylated Cas through SH2 and SH3 domain-binding motifs in the Cas C-terminal region (37Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar); and the protein-tyrosine phosphatase PTP-PEST (38Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar). Cas also associates with the focal adhesion proteins paxillin and tensin (39Salgia R. Pisick E. Sattler M. Li J.-L. Uemura N. Wong W.K. Burky S. Hirai H. Chen L.B. Griffin J.D. J. Biol. Chem. 1996; 271: 25198-25203Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Binding of Crk family members to tyrosine-phosphorylated Cas illustrates the assembly of signaling complexes since the SH3 domain of Crk proteins can bind in turn to a number of proteins, including two guanine nucleotide exchange factors, Sos and C3G, which regulate Ras and Rap1 activation, respectively (40Feller S. Knudsen B. Hanafusa H. Oncogene. 1995; 10: 1465-1473PubMed Google Scholar, 41Matsuda M. Hashimoto K. Muroya K. Hasegawa H. Kurata T. Tanaka S. Nakamura S. Hattori S. Mol. Cell. Biol. 1994; 14: 5495-5500Crossref PubMed Scopus (183) Google Scholar, 42Knudsen B.S. Feller S.M. Hanafusa H. J. Biol. Chem. 1994; 269: 32781-32787Abstract Full Text PDF PubMed Google Scholar, 43Smit L. van der Horst G. 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To date, the identification of Cas-associated Src kinases in nontransfected cells is not well documented. In this study, we present evidence that in the human multiple myeloma cell line ARH-77, p59Fyn tyrosine kinase (Fyn) and SHP2 tyrosine phosphatase are recruited to the Cas complex following integrin ligation. This study provides insight into the control of integrin-mediated tyrosine phosphorylation of Cas in B cells. ARH-77 cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum. Antibodies used in this study were directed against the following: CD29/β1 integrin (mAb 1The abbreviations used are: mAb, monoclonal antibody; GST, glutathione S-transferase; HA, hemagglutinin. K20, provided by Prof. Alain Bernard, U146 INSERM, Nice, France), phosphotyrosine (mAb 4G10), p59Fyn or p130Cas (Cas) (rabbit polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA), GST (GST mAb, Santa Cruz Biotechnology, Santa Cruz, CA), SHP2 (PTP-1D mAb, Transduction Laboratories, Lexington, KY), hemagglutinin (HA mAb, Boehringer Mannheim), and affinity-purified rabbit anti-mouse Ig (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). GST-Fyn fusion proteins were provided by Drs. Joan Brugge and Martyn Botfield (Ariad Pharmaceuticals, Cambridge, MA). ARH-77 cells were stimulated with anti-integrin antibodies plus rabbit anti-mouse Ig as described previously (14Manié S. Astier A. Wang D. Phifer J. Chen J. Lazarovits A. Morimoto C. Freedman A. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar). Cells were then lysed in 0.5% Nonidet P-40 buffer containing 150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 mm NaF, and 1 mmsodium vanadate. Fyn cDNA inserted into the pSRα2 expression vector was provided by Dr. Antonio Da Silva (Dana-Farber Cancer Institute) (45Da Silva A. Rosenfield J. Mueller I. Bouton A. Hirai H. Rudd C. J. Immunol. 1997; 158: 2007-2016PubMed Google Scholar). Rat Cas cDNA and deletion mutants of Cas cDNA were cloned into a modified version of the pcDL-SRα296 expression plasmid, termed pSP65-SRα.2-HAtag-Hygro, containing a hygromycin B phosphotransferase gene and an HA epitope tag sequence in frame with the Cas cDNAs (46Pulido R. Schlossman S.F. Saito H. Streuli M. J. Exp. Med. 1994; 179: 1035-1040Crossref PubMed Scopus (15) Google Scholar, 47Serra-Pages C. Kedersha N. Fazikas L. Medley Q. Debant A. Streuli M. EMBO J. 1995; 14: 2827-2838Crossref PubMed Scopus (289) Google Scholar). Briefly, 5′-fragments of the cDNAs with an in-frame XbaI site were generated using polymerase chain reaction and cloned together with the corresponding 3′-fragment into the restriction sites XbaI andEcoRI of pSP65-SRα.2-HAtag-Hygro. The expression plasmids were used to transiently transfect COS-7 cells by the DEAE-dextran/Me2SO method as described (48Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). For immunoprecipitation studies, cell lysates were precleared with protein G-Sepharose beads (Pharmacia, Uppsala) and then preincubated with specific antibody for 1 h at 4 °C, followed by the addition of protein G-Sepharose beads for 1 h at 4 °C. For precipitations with GST fusion proteins, lysates were incubated for 2 h at 4 °C with 25 μg of fusion proteins bound to glutathione beads (Pharmacia). Precipitated proteins were washed four times with lysis buffer and subjected to kinase assay or eluted by boiling in sample buffer (2% SDS, 10% glycerol, 0.1 m Tris, pH 6.8, 0.02% bromphenol blue). For sequential immunoprecipitation, washed beads were boiled for 5 min in the presence of 2% SDS, and the supernatants were reprecipitated with antibodies in lysis buffer containing a 0.1% final SDS concentration. In vitro kinase assays were performed by washing Cas immunoprecipitates once in kinase buffer (10 mmHepes, pH 7.3, containing 50 mm NaCl, 5 mmMnCl2, 5 mm MgCl2, and 100 μm sodium vanadate) and incubating the pellet in kinase buffer containing 0.1 mm ATP (Sigma) for 10 min at room temperature. Proteins were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to Immobilon-P™ membranes (Millipore Corp., Bedford, MA). Membranes were blocked using 5% nonfat dried milk in TBS-T (20 mm Tris, pH 7.6, 130 mm NaCl, 0.1% Tween 20) and incubated for 1 h with specific antibodies in TBS-T. Immunoreactive bands were visualized using secondary horseradish peroxidase-conjugated antibodies (Promega, Madison, WI) and chemiluminescence (ECL, Amersham International, Buckinghamshire, United Kingdom). To examine Cas-associated tyrosine kinase(s), the B cell line ARH-77 was stimulated with the anti-β1 integrin mAb K20 followed by rabbit anti-mouse Ig. Cell lysates were then immunoprecipitated with anti-Cas antibody, and subjected (+) or not (−) to an in vitrokinase assay for 10 min (Fig. 1 A, upper panels). Anti-phosphotyrosine Western blot analysis showed that Cas immune complexes contained a transient tyrosine kinase(s) activity (maximally detected at 5 and 15 min), which resulted in increasedin vitro tyrosine phosphorylation of Cas plus an additional main band ranging from 55 to 60 kDa. The same membrane was reprobed with anti-Cas antibody to show that equivalent amounts of immunoprecipitated Cas were loaded in each lane (Fig. 1 A,middle panels). The in vitro phosphorylated 55–60-kDa band contained a sharp 59-kDa protein, which was suggestive of the presence of p59Fyn kinase. To investigate this possibility, the membrane was reprobed with anti-p59Fynantibody. As shown in Fig. 1 A, (lower panels), anti-Fyn antibody reacted with a faint band detectable only in the5 and 15 min lanes of stimulation. To further confirm the identity of this 59-kDa protein, anti-Cas or control (Ct) immunoprecipitates from 15-min β1 integrin-stimulated ARH-77 cells were subjected to in vitro kinase assays (Ip 1), and half of the samples were then reimmunoprecipitated with anti-Fyn antibody (Ip 2). As shown in Fig. 1 B (lower panel), Fyn could be reimmunoprecipitated from Cas immune complexes, but not from control immunoprecipitates. Fyn was tyrosine-phosphorylated (Fig.1 B, upper panel) and comigrated with the sharp pp59. These results indicate that Fyn kinase associates in an integrin-regulated manner with Cas and strongly suggest that it participates in Cas phosphorylation. The C terminus of Cas contains a proline-rich sequence (RPLPSPP) and a YDYV motif, which have been shown to bind to Src SH3 and SH2 domains, respectively (37Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Since we have previously reported that a GST fusion protein containing the RPLPSPP sequence bound to Fyn (24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), we further examined the requirement of this region of Cas for Fyn/Cas interaction. cDNAs encoding HA-tagged wild-type Cas or deletion mutants of Cas were transiently expressed in COS-7 cells in the presence or absence of cDNA encoding Fyn. The mutants of Cas included the following: Cas ΔSD, in which the sequence from amino acids 213 to 514, which contains the substratedomain with 15 out of 27 potential sites of tyrosine phosphorylation within Cas, was deleted; and Cas ΔSB, in which the sequence from amino acids 638 to 889, which contains the Src SH2 and SH3 domain-binding motifs, was deleted (37Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) (Fig. 2). To discriminate transfected Cas proteins from endogenous Cas proteins, lysates of transfected COS-7 cells were immunoprecipitated with anti-HA tag antibody. Anti-phosphotyrosine immunoblotting of the anti-HA tag immunoprecipitations indicated that the presence of cotransfected Fyn led to Cas phosphorylation (Fig. 3 A). The Cas ΔSD mutant, although lacking some of the potential sites of tyrosine phosphorylation, showed increased tyrosine phosphorylation in the presence of Fyn. In contrast, the Cas ΔSB mutant cotransfected with Fyn demonstrated reduced tyrosine phosphorylation when compared with Cas or Cas ΔSD. Therefore, the SB domain of Cas was necessary for Fyn-mediated Cas phosphorylation. Comparable levels of expression of Cas, Cas mutants (Fig. 3 B), and Fyn (Fig. 3 D) were detected regardless of the cotransfection conditions. More important, Fyn kinase was co-immunoprecipitated with Cas and Cas ΔSD, but not with Cas ΔSB (Fig. 3 C), indicating that the SB region was required for a physical interaction between Fyn and Cas.Figure 3Tyrosine phosphorylation of Cas by coexpression with Fyn kinase in COS-7 cells requires the Src-binding site of Cas. COS-7 cells were transiently transfected with control plasmid (Mock) or with plasmid encoding HA-tagged wild-type Cas (Cas WT), HA-tagged Cas with the Src-binding site deleted (ΔSB), or HA-tagged Cas with the substrate domain deleted (ΔSD). A second set of cells were also cotransfected with a plasmid encoding Fyn (+ Fyn). Transfected Cas was immunoprecipitated (Ip) using anti-HA antibodies and analyzed by Western blotting with anti-Tyr(P) antibody (P-Tyr) (A). The positions of Cas, Cas ΔSB, Cas ΔSD, and Fyn are indicated. The membrane was then stripped and reblotted with anti-Cas (Cas; B) or anti-Fyn (Fyn; C) antibodies as indicated. Cotransfected cell lysates were also immunoprecipitated with anti-Fyn antibodies to control for the amount of Fyn expression within the cells (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further study the interaction between Cas and Fyn, we performed binding experiments with truncated GST-Fyn fusion proteins corresponding to the SH2 or SH3 domain of Fyn. Fusion proteins were incubated with lysates of unstimulated (Fig. 4 A, 0 lanes) or β1 integrin-stimulated (β1 lanes) ARH-77 cells, and the presence of Cas was analyzed by immunoblotting. To control for the integrin-mediated Cas tyrosine phosphorylation, anti-phosphotyrosine immunoprecipitations from the same samples were also analyzed. Fig.4 A shows that Cas did not bind to GST protein alone. In contrast, Cas that was derived from stimulated cells was able to bind to the GST-Fyn SH2 domain, whereas Cas from both unstimulated and stimulated cells bound to the GST-Fyn SH3 domain. In this experiment, Cas was seen to migrate as 105- and 130-kDa bands as described previously (24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), with the main increased phosphorylation in the 130-kDa form. Hyperphosphorylated Cas precipitated with anti-phosphotyrosine antibody or GST-Fyn SH2 protein resolved with a slower migration (27Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). The membrane was reprobed with anti-GST antibody to show that comparable amounts of GST fusion proteins were used to precipitate Cas (Fig. 4 B). These results indicate that the Fyn SH3 domain binds to Cas in vitro and that integrin-stimulated phosphorylation of Cas creates a binding site for the Fyn SH2 domain. To investigate the presence of a Cas-associated tyrosine phosphatase, Cas immunoprecipitates from β1 integrin-stimulated ARH-77 cells were subjected to an in vitro kinase assay with or without the tyrosine phosphatase inhibitor sodium vanadate (Fig. 5 A). The tyrosine phosphorylation of Cas was markedly increased with the addition of sodium vanadate in the kinase assay. These results indicate that a tyrosine phosphatase activity is associated with Cas immunoprecipitates. Because the tyrosine phosphatase SHP2 has been shown recently to associate through its SH2 domains with the Cas-related Cas-L (HEF1) protein (25Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (146) Google Scholar), we tested whether SHP2 also associated with Cas. Unstimulated or β1 integrin-stimulated ARH-77 cells were immunoprecipitated with anti-Cas (Cas) or control (Ct) antibodies and immunoblotted with anti-SHP2 antibody (Fig. 5 B). Compared with unstimulated cells and control immunoprecipitates, a 72-kDa band reactive with anti-SHP2 antibody was clearly increased in Cas complexes isolated from β1 integrin-stimulated cells. ARH-77 cells were then β1 integrin-stimulated for 0, 2, 5, and 15 min, and cell lysates were immunoprecipitated with anti-Cas antibody (Fig. 5 C). Cas quantification was comparable at 2, 5, and 15 min and showed increased tyrosine phosphorylation, which correlated with Cas-SHP2 complex formation, which was increased at the 5- and 15-min time points. These results suggest that similar to HEF1/Cas-L, SHP2 associates with Cas in an integrin-regulated manner. Therefore, SHP2 is likely to be associated with the phosphatase activity observed in vitro and might play a role in the in vivo control of Cas dephosphorylation. Integrins are involved in the regulation of proliferation, differentiation, and cell survival in a variety of cell types, events that are dependent upon tyrosine phosphorylation (49Schwartz M. Cancer Res. 1993; 53: 1503-1506PubMed Google Scholar). Cas has been identified to be a major tyrosine-phosphorylated substrate following integrin ligation (20Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 21Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 22Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 23Petruzzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In this study, we found that precipitations of Cas immune complexes from integrin-stimulated cells contained both tyrosine kinase and tyrosine phosphatase activities, which modulated the in vitro phosphorylation of Cas. Furthermore, Fyn tyrosine kinase and SHP2 tyrosine phosphatase were recruited in Cas complexes, suggesting that they participate in modulating Cas phosphorylation. The C-terminal proline-rich region of Cas can associate in vitro with several Src kinases, including p59Fyn, p59/62Hck, and p53/56Lyn (24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In this study, we found that in ARH-77 cells, Fyn was the most obvious kinase detectable. The focal adhesion kinases FAK and RAFTK can associate with Cas (18Astier A. Avraham H. Manié S.N Groopman J. Canty T. Avraham S. Freedman A.S. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 31Polte T. Hanks S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (384) Google Scholar, 32Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 36Burnham M. Harte M. Richardson A. Parsons J. Bouton A. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar). In B cells, RAFTK and, to a lesser extent, FAK are both tyrosine-phosphorylated following integrin ligation (14Manié S. Astier A. Wang D. Phifer J. Chen J. Lazarovits A. Morimoto C. Freedman A. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar, 18Astier A. Avraham H. Manié S.N Groopman J. Canty T. Avraham S. Freedman A.S. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Similar to Src kinases, the kinase activity of RAFTK and FAK correlates with an increase in autophosphorylation activity in certain cell types (15Avraham S. London R. Fu Y. Ota S. Hiregowdara D. Li J. Jiang S. Pasztor L.M. White R.A. Groopman J.E. Avraham H. J. Biol. Chem. 1995; 270: 27742-27751Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 50Hildebrand J. Schaller M. Parsons J. J. Cell Biol. 1993; 123: 993-1005Crossref PubMed Scopus (356) Google Scholar). However, we previously reported that Cas associated with RAFTK is mainly nonphosphorylated on tyrosine residues (18Astier A. Avraham H. Manié S.N Groopman J. Canty T. Avraham S. Freedman A.S. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). In addition, we did not observe tyrosine-phosphorylated bands corresponding in size to RAFTK and FAK, 120 and 125 kDa, respectively. This results suggest that in nontransfected cells and under the conditions of these assays, these two kinases may not be sufficient for Cas phosphorylation. Further support of this is the observation that Cas phosphorylation is reduced in fibroblasts lacking Src kinases, but remains unaffected in fibroblasts lacking FAK (34Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar, 35Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar). In a cotransfection assay in COS-7 cells, we found that the Fyn/Cas physical interaction and subsequent Fyn-mediated Cas phosphorylation required amino acids 638–889 in the C-terminal region of Cas. This sequence contains a polyproline stretch (RPLPSPP) and a YDYV motif to which both Fyn SH3 and SH2 domains could bind in vivo.In vitro studies using GST fusion proteins derived from the SH2 and SH3 domains of Fyn indicated that both domains could participate in this Fyn/Cas interaction. However, the in vivo association of Fyn and Cas was transient and correlated with Cas tyrosine phosphorylation, implying a regulation of this interaction. Whether the Fyn SH3 domain is primarily involved in Cas binding or is subsequent to Fyn SH2 binding to reinforce the interaction between the two proteins is not known. A precedent for the predominant binding of the SH2 domain over the SH3 domain has been described for the interaction of p120Cbl with phosphoinositol 3′-kinase (51Fukazawa T. Reedquist K.A. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S.E. Band H. J. Biol. Chem. 1995; 270: 19141-19150Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The Cas-like protein Sin leads to activation of Src kinase activity on binding of Sin to the c-Src SH3 domain (30Alexandropoulos K. Baltimore D. Genes Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar). Similarly, it is possible that Fyn binding to Cas will stimulate the kinase activity of Fyn, which in turn will lead to the effective phosphorylation of Cas and allow the recruitment of SH2 domain-containing molecules such as the adapter proteins of the Crk family (23Petruzzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 2Astier, A., Manié, S. N., Law, S. F., Canty, T., Hagheyeghi, N., Druker, B. J., Salgia, R., Golemis, E. A., and Freedman, A. S. (1997) Leukemia Lymphoma, in press. In support of this is the finding that Fyn-phosphorylated Cas in COS cells interacts with Crk (data not shown). The assembly of this complex of proteins might further enable recruited proteins to interact with and potentially be phosphorylated by Fyn. Since the phosphorylation of Cas is likely to be an important event in the propagation and amplification of downstream signals of integrin ligation, a mechanism of down-regulating that process is critical. A role for SHP2 tyrosine phosphatase in modulating the phosphorylation of Cas in B cells came from the finding that SHP2 was recruited in Cas complexes following integrin ligation. The Cas-SHP2 association correlated with the extent of Cas tyrosine phosphorylation. In view of the recent finding that SHP2 associates with HEF1/Cas-L through its SH2 domains (25Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (146) Google Scholar), it is possible that a similar mechanism occurs for Cas-SHP2 assembly. This might result in activation of SHP2 activity since engagement of its SH2 domains following binding to platelet-derived growth factor receptor β stimulates its tyrosine phosphatase activity (52Lechleider R.J. Sugimoto S. Bennett A.M. Kashishian A.S. Cooper J.A. Shoelson S.E. Walsh C.T. Neel B.G. J. Biol. Chem. 1993; 268: 21478-21481Abstract Full Text PDF PubMed Google Scholar). Two other protein-tyrosine phosphatases, namely PTP-1B and PTP-PEST, can also associate with Cas (33Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 38Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar) and might participate in the Cas-associated phosphatase activity. The precise mechanism by which integrin cross-linking leads to the activation of the kinase(s) and phosphatase(s) is presently unclear. Integrin-mediated phosphorylated Cas localizes to focal adhesions, whereas nonphosphorylated Cas remains in the cytosol (22Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 27Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). Such Cas localization may be driven by its constitutive association through its SH3 domain with FAK, due to the FAK C-terminal focal adhesion targeting domain (50Hildebrand J. Schaller M. Parsons J. J. Cell Biol. 1993; 123: 993-1005Crossref PubMed Scopus (356) Google Scholar). Therefore, redistributed Cas during integrin stimulation might localize Cas to a region of the cell where Cas will be accessible to tyrosine kinases. Support for this hypothesis is that optimal Cas phosphorylation requires an intact cytoskeleton since inhibitors of cytoskeletal assembly also inhibit integrin-mediated Cas tyrosine phosphorylations (20Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). A potential sequence of events leading to Cas phosphorylation in ARH-77 cells could be that integrin-mediated cytoskeletal reorganization allows the co-compartmentalization of Cas and Fyn. Activated Fyn, possibly by its binding to Cas, phosphorylates Cas, leading to the formation of a signaling complex including Crk family members and SHP2. SHP2, which becomes activated following SH2-mediated binding to phosphorylated Cas, could then participate in downstream signaling events and/or attenuate the activity of the complex by dephosphorylating Cas. Cas and the Cas-like protein HEF1 are both expressed in normal B cells and most B cell lines (24Manié S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). However, Cas is the predominant substrate that is phosphorylated in terminally differentiated B cell lines following β1 integrin ligation, whereas HEF1 is the major phosphorylated substrate in normal tonsillar B cells and other B cell lines. It is unknown why Cas or HEF1 is the favored substrate in certain cells, although this may be related to the compartmentalization and activation of specific kinases during integrin-induced cytoskeleton reorganization. We thank Prof. Alain Bernard for K20 antibody, Drs. Joan Brugge and Martyn Botfield for Fyn constructs, and Dr. Antonio Da Silva for providing Fyn cDNA. We greatly appreciate Dr. Andreas Beck for help in preparing Cas constructs. We also thank Drs. Andreas Beck, Antonio Da Silva, Bernard Mari, Susan Law, and Erica Golemis for helpful discussions. We thank Janet Walsh for assistance in preparing the manuscript.

CrkL, a cellular homologue of the v-crk oncogene, belongs to the family of adaptor proteins, containing SH2 and SH3 domains, but no catalytic domain. Stimulation of normal B-cells and B-cell lines through β1 integrin or - cell antigen receptor (BCR) promoted the association of CrkL with a set of 105-130 kD tyrosine phosphorylated substrates. The principal substrate is a recently identified molecule known as p105HEFI (HEF1), which is highly homologous to p130Cas (Cas), the major tyrosine-phosphorylated protein detected in fibroblasts after transformation by v-crk. Immunodepletion studies indicated that all the tyrosine phosphorylated HEF1 or Cas was complexed with CrkL. Furthermore, the guanine nucleotide exchange factor C3G, which is thought to be involved in the regulation of the ras pathway and constituvely binds to the C-terminal SH3 domain of CrkL, could be detected in HEF1 immunoprecipitates. Therefore, CrkL is involved in the formation of a HEFl-CrkL-C3G ternary complex in B-cells, suggesting that it is likely to play an important role, allowing the propagation of the stimulation initiated by both BCR and β1 integrin ligation.

The production of interleukin 1 (IL1), a pleiotropic monocyte-derived interleukin, can be induced in vitro by various stimuli. The present study shows that cytochalasins which inhibit actin filament polymerization in various cell types have no significant effect on IL1 production from human monocytic cells. On the contrary, microtubule disrupters such as colchicine, vinblastine, and vincristine dramatically potentiate (15- to 35-fold), in a dose-dependent fashion, cell-associated IL1 and to a lesser extent (2.5- to 7-fold) released IL1 in the myelomonocytic THP1 cell line and in adherent peripheral blood mononuclear cells. The enhancing effect of the drugs was blocked by actinomycin D and by cycloheximide and was accompanied by an increase of specific IL1 β mRNA expression as measured by Northern blot analysis, thus indicating that these drugs act at a transcriptional or post-transcriptional IL1 gene expression level.

Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.

Both the hexosamine biosynthetic pathway (HBP) and the endoplasmic reticulum (ER) are considered sensors for the nutritional state of the cell. The former is a branch of the glucose metabolic pathway that provides donor molecules for glycosylation processes, whereas the second requires co-translational N-glycosylation to ensure proper protein folding. It has become clear that the microenvironment of solid tumours, characterised by poor oxygen and nutrient supply, challenges optimal functions of the ER and the HBP. Here, we review recent advances demonstrating that the ER stress (ERS) response and HBP pathways are interconnected to promote cell viability. We then develop the idea that communication between ER and HBP is a survival feature of neoplastic cells that plays a prominent role during tumourigenesis.

Abstract B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105–130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105–125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins.

Background Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes–associated protein whose expression level affects cellular free cholesterol content. Methodology/Principal Findings Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY. Conclusions/Significance LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.

Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation.

Abstract Purpose: Multiple myeloma is a clonal plasma cell disorder in which growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type I (IGF-I). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma. Experimental Design: We analyzed the impact of IGF-I combined with proteasome inhibitors on multiple myeloma cell lines in vivo and in vitro as well as on fresh human myeloma cells. Results: Our study shows that IGF-I enhances the cytotoxic effect of proteasome inhibitors against myeloma cells. The effect of bortezomib on the content of proapoptotic proteins such as Bax, Bad, Bak, and BimS and antiapoptotic proteins such as Bcl-2, Bcl-XL, XIAP, Bfl-1, and survivin was enhanced by IGF-I. The addition of IGF-I to bortezomib had a minor effect on NF-κB signaling in MM.1S cells while strongly enhancing reticulum stress. This resulted in an unfolded protein response (UPR), which was required for the potentiating effect of IGF-I on bortezomib cytotoxicity as shown by siRNA-mediated inhibition of GADD153 expression. Conclusions: These results suggest that the high baseline level of protein synthesis in myeloma can be exploited therapeutically by combining proteasome inhibitors with IGF-I, which possesses a “priming” effect on myeloma cells for this family of compounds. Clin Cancer Res; 19(13); 3556–66. ©2013 AACR.

Most Toll-like receptors and IL-1/IL-18 receptors activate a signaling cascade via the adaptor molecule MyD88, resulting in NF-κB activation and inflammatory cytokine and chemokine production. Females are less susceptible than males to inflammatory conditions, presumably due to protection by estrogen. The exact mechanism underlying this protection is unknown.MCF7 cells expressing wild-type or mutated LXXLL motif were used to determine MyD88/estrogen receptor (ER)-a interaction by immunoprecipitation and cell activation by ELISA and luciferase reporter assay. IL-1b and/or E2 were used to activate MCF7 cells expressing normal or knocked down levels of PRMT1. Finally, in situ proximity ligation assay with anti-MyD88 and anti-methylated ER-a (methER-a) antibodies was used to evaluate MyD88/methylated ER-a interaction in THP1 cells and histological sections.We show that MyD88 interacts with a methylated, cytoplasmic form of estrogen receptor-alpha (methER-α). This interaction is required for NF-κB transcriptional activity and pro-inflammatory cytokine production, and is dissociated by estrogen. Importantly, we show a strong gender segregation in gametogenic reproductive organs, with MyD88/methER-α interactions found in testicular tissues and in ovarian tissues from menopausal women, but not in ovaries from women age 49 and less - suggesting a role for estrogen in disrupting this complex in situ.Collectively, our results indicate that the formation of MyD88/methER-α complexes during inflammatory signaling and their disruption by estrogen may represent a mechanism that contributes to gender bias in inflammatory responses.

mTOR and Unfolded Protein Response (UPR) are two signaling pathways frequently activated in cancer cells. The mTOR pathway has been shown to be up-regulated in most gastroenteropancreatic neuroendocrine tumors. In contrast, little is known about the UPR status in neoplastic neuroendocrine cells. However, these hormone-producing cells are likely to present distinctive adaptations of this pathway, as other secretory cells. We therefore analyzed the status of the three axes of UPR and their relation to mTOR pathway in two gastrointestinal neuroendocrine tumors (GI-NET) cell lines STC-1 and GluTag. At baseline, pharmacological inducers activate the three arms of UPR: PERK, ATF6 and IRE1. Although hypoxia stimulates the PERK, ATF6 and IRE-1 pathways in both cell lines, glucose depletion activates UPR only in STC-1 cell line. Strikingly, P-p70S6K1 increases concomitantly to P-PERK and BiP in response to thapsigargin treatment, glucose depletion or hypoxia. We found that different mTOR inhibitors activate the PERK signaling pathway. To confirm that mTOR inhibition modulates PERK activation, we inhibited PERK and showed that it decreased cell viability when associated to mTOR inhibition, indicating that mTOR drives a PERK-dependent survival pathway. In conclusion, in GI-NET cell lines, UPR signaling is functional and PERK arm is induced by mTOR inhibition. These observations open up new perspectives for therapeutic strategies: the crosstalk between mTOR and UPR might contribute to the resistance to mTOR inhibitors and could be targeted by mTOR and PERK inhibitors in combination therapy.

The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment.

Infection of mouse L.CD46 fibroblasts with measles virus resulted in a poor virus yield, although no defects in the steps of virus binding, entry or fusion, were detected. Two days post-infection, the level of expression of the viral F protein was found to be similar on the surface of infected L.CD46 and HeLa cells using a virus multiplicity enabling an equal number of cells to be infected. After immunofluorescence labelling and confocal microscopy, L.CD46 cells also displayed a significant increase in the co-localisation of the N protein with the cell surface H and F proteins. Immunogold labelling and transmission electron microscopy demonstrated the accumulation of numerous nucleocapsids near the plasma membrane of L.CD46 cells with little virus budding, in contrast to infected HeLa cells which displayed fewer cortical nucleocapsids and more enveloped viral particles. Purified virus particles from infected L.CD46 contained a reduced amount of H, F and M protein. Altogether, these data indicate that, in L.CD46 cells, the late stage of measles virus assembly is defective. This cellular model will be helpful for the identification of cellular factors controlling measles virus maturation.

Abstract Germline RET mutations are responsible for different inherited disorders: Hirschsprung disease (congenital aganglionic megacolon), caused by loss of function mutations, familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2, caused by gain of function mutations. Intriguingly, some RET mutations, including C620R, are associated with both types of diseases. To investigate the dual role of such RET mutations, a mouse model with a targeted mutation ret C620R was generated. ret C620R/C620R offspring die during the first postnatal day, and show kidney agenesis and intestinal aganglionosis. Decreased outgrowth of the Ret‐positive cells was observed in ret C620R/C620R neuronal cell cultures, which is suggestive of an impaired migration, proliferation or survival of the Ret‐expressing cells. Electronmicroscopy revealed the absence of membrane‐bound Ret in ret C620R/C620R cells as compared to ret +/+ and ret +/C620R cells. On the other hand, aged ret +/C620R mice develop precancerous lesions in the adrenal gland or in the thyroid. Our results suggest that the ret C620R mutation has a loss of function effect in homozygotes and exhibits a dominant gain of function effect with low penetrance causing hyperplasia in heterozygotes. © 2007 Wiley‐Liss, Inc.

We had previously reported that the CD29 mAb "K20," presented in a soluble form, blocks peripheral T cell proliferation/activation induced by a CD3 mAb. To better characterize the negative signal delivered by soluble K20, we have investigated its effects on the phospholipid metabolism, both in Jurkat and CD4+ T cells. In CD3-activated T cells, K20 inhibited the increase of diacylglycerol (DAG) and phosphatidic acid levels, but did not modify phosphatidylinositol 4,5-bisphosphate levels, cytosolic Ca2+ raise, and inositolphosphates formation, indicating that K20 did not inhibit phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C-gamma. Moreover, in these conditions, K20 increased phosphatidylethanolamine levels, without variation of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, suggesting that K20 specifically increased the phosphatidylethanolamine biosynthesis from DAG. Thus, the effects of K20 on DAG and phosphatidic acid levels resulted from an accelerated catabolism rather than from a defect of synthesis. That K20 acts solely at an early step of T cell activation, namely before the binding of IL-2 to its receptor, is supported by the observation that adding exogenous rIL-2 increased proliferation in spite of K20. These results suggest that the beta 1 integrin molecules interact with the membrane phospholipid metabolism and they appear to be the hallmark of a peculiar negative pathway of T cell activation, likely to play an important regulatory role mediated via the T cell integrin molecules.

In this study we investigated the T cell signals required for monocyte activation. We used an in vitro co-culture system involving two human cell lines: Jurkat T cells and THP-1 monocytes. Monocyte activation was monitored by measuring IL-1 beta production, whereas IL-2 secretion reflected Jurkat activation. We showed that CD-3 -stimulated Jurkat cells delivered an IL-1-inductive signal to THP-1 cells through a cellular contact which was independent of THP-1 Fc receptors cross-linking. Stimulation of IL-1 beta production did not appear to require lymphokine secretion by T cell since a lymphokine defective mutant of Jurkat cell was able to deliver the stimulatory signal. The LFA-1 molecule was clearly shown to participate in the cooperation process, but its role was likely to be restricted to mediating initial adhesive interaction rather than to transducing the IL-1 -inductive signal. Interestingly, the co-culture stimulated by (Fab')2 fragments of CD3 mAb displayed an enhanced IL-1 beta production without any increase of IL-2 secretion. This result indicated that Jurkat cells could stimulate THP-1 cells even when they were only partially activated. The kinetics and conditions of IL-1 beta production called our attention to the early T cell activation antigen CD69. We then showed that CD69 mAb interfered with transmission of the IL-1 inductive signal (40-50% inhibition of IL-1 production). Our results are suggestive of a new role for CD69 molecule intervening in the T lymphocyte-dependent monocyte activation process.

Experiments were performed on blood samples from 5 cosmonauts in order to investigate the effects of long duration spaceflight (26 to 166 days) on immune activity. The experiments were performed on cultured mononuclear cells purified from blood samples collected during the preflight period and 24 h after landing. The production of interleukin 2, which is the major cytokine involved in T lymphocyte proliferation, was found to be enhanced after flight in some individuals, whereas the ability of mitogen-stimulated cells to express interleukin 2 receptor was impaired 24 h after flight for two cosmonauts out of five. Normal interleukin 2 receptor expression was obtained in all cases when lymphocytes were directly activated by a protein kinase C activating phorbol ester. On the other hand, no significant changes were observed in interleukin 1 production by cultured peripheral blood mononuclear cells. Lastly, the distribution of T lymphocytes subsets was examined in peripheral blood sampled 24 h after landing and was found to be within normal values.

Studies of the topology, functioning, and regulation of metabolic systems are based on two main types of information that can be measured by mass spectrometry: the (absolute or relative) concentration of metabolites and their isotope incorporation in 13C-labeling experiments. These data are currently obtained from two independent experiments because the 13C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the 13C-mass fractions from which its 13C-isotopologue distribution (CID) is quantified. Here, we developed a generic method with a dedicated processing workflow to obtain these two sets of information simultaneously in a unique sample collected from a single cultivation, thereby reducing by a factor of 2 both the number of cultivations to perform and the number of samples to collect, prepare, and analyze. The proposed approach is based on an IS labeled with other isotope(s) that can be resolved from the 13C-mass fractions of interest. As proof-of-principle, we analyzed amino acids using a doubly labeled 15N13C-cell extract as IS. Extensive evaluation of the proposed approach shows a similar accuracy and precision compared to state-of-the-art approaches. We demonstrate the value of this approach by investigating the dynamic response of amino acids metabolism in mammalian cells upon activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a key component of the unfolded protein response. Integration of metabolite concentrations and isotopic profiles reveals a reduced de novo biosynthesis of amino acids upon PERK activation. The proposed approach is generic and can be applied to other (micro)organisms, analytical platforms, isotopic tracers, or classes of metabolites.

Advances in cancer biology over the past decades have revealed that metabolic adaptation of cancer cells is an essential aspect of tumorigenesis. However, recent insights into tumour metabolism in vivo have revealed dissimilarities with results obtained in vitro. This is partly due to the reductionism of in vitro cancer models that struggle to reproduce the complexity of tumour tissues. This review describes some of the discrepancies in cancer cell metabolism between in vitro and in vivo conditions, and presents current methodological approaches and tools used to bridge the gap with the clinically relevant microenvironment. As such, these approaches should generate new knowledge that could be more effectively translated into therapeutic opportunities.

Integrin crosslinking on human B cells induces tyrosine phosphorylation of a set of proteins ranging from 105 to 130 kDa, among which is the focal adhesion kinase p125FAK. Here we show that the c-CBL protooncogene product p120c-CBL is a component of these substrates. beta 1 integrin stimulation of p120c-CBL phosphorylation was observed in both transformed and normal human B cells, and was inhibited by prior treatment of cells with cytochalasin B, which disrupts the actin network. In contrast, tyrosine phosphorylation of p120c-CBL following crosslinking of the B cell antigen receptor (BCR) was not affected by cytochalasin B. Integrin stimulation of the promegakaryocytic cell line MO7e also led to a cytoskeleton-dependent tyrosine phosphorylation of p120c-CBL. In MO7e cells, this stimulation was induced by ligation of either beta 1 or beta 2 integrin, whereas only by ligation of beta 1 integrin in B cells. Tyrosine phosphorylation of p120c-CBL links phosphatidylinositol-3 kinase (PI-3K) with the BCR signaling machinery. Although the p85 subunit of PI-3K was increased in p120c-CBL immunoprecipitates from BCR-stimulated B cells, this association was only minimally increased by beta 1 integrin ligation. The function of p120c-CBL remains unknown; however, its interactions in vitro and in vivo with Src homology 2 and 3 (SH2 and SH3) domain-containing proteins suggest that p120c-CBL has a significant function in signal transduction pathways, and therefore may play a role in integrin signaling in lymphoid and hematopoietic cells.

Genetic alterations in non-small cell lung cancers (NSCLC) stimulate the generation of energy and biomass to promote tumor development. However, the efficacy of the translation process is finely regulated by stress sensors, themselves often controlled by nutrient availability and chemotoxic agents. Yet, the crosstalk between therapeutic treatment and glucose availability on cell mass generation remains understudied. Herein, we investigated the impact of pemetrexed (PEM) treatment, a first-line agent for NSCLC, on protein synthesis, depending on high or low glucose availability. PEM treatment drastically repressed cell mass and translation when glucose was abundant. Surprisingly, inhibition of protein synthesis caused by low glucose levels was partially dampened upon co-treatment with PEM. Moreover, PEM counteracted the elevation of the endoplasmic reticulum stress (ERS) signal produced upon low glucose availability, providing a molecular explanation for the differential impact of the drug on translation according to glucose levels. Collectively, these data indicate that the ERS constitutes a molecular crosstalk between microenvironmental stressors, contributing to translation reprogramming and proteostasis plasticity.

Abstract Most Toll-like receptors and IL-1/IL-18 receptors activate a signaling cascade via the adaptor molecule MyD88, resulting in NF-κB activation and inflammatory cytokine and chemokine production. Females are less susceptible than males to inflammatory conditions, presumably due to protection by estrogen. Here we show that MyD88 interacts with a methylated, cytoplasmic form of estrogen receptor-alpha (methER-α). This interaction is required for NF-κB transcriptional activity and pro-inflammatory cytokine production, and is dissociated by estrogen. Importantly, we show a strong gender segregation in gametogenic reproductive organs, with MyD88/methER-α interactions found in testicular tissues and in ovarian tissues from menopausal women, but not in ovaries from women age 49 and less -suggesting a role for estrogen in disrupting this complex in situ . Collectively, our results indicate that the formation of MyD88/methER-α complexes during inflammatory signaling and their disruption by estrogen may represent a mechanism that contributes to gender bias in inflammatory responses.

Abstract Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor GCN2 represses the expression of the precursor of ribosomal RNA, 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, GCN2 activity supports cell proliferation through the transcription stimulation of 47S rRNA, independently of the canonical ISR axis. However, impairment of GCN2 activity prevents nuclear translocation of the methionyl tRNA synthetase (MetRS) underlying the generation of a nucleolar stress, mTORC1 inhibition and autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA pol I inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls the ribosome biogenesis according the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and the RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically-stressed COAD cell.

&lt;p&gt;PDF file - 175K, CD44-ICD-CREB complex in human PTC cell lines&lt;/p&gt;

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&lt;p&gt;PDF file - 78K, CD44-ICD expression in human thyroid cancer cell lines&lt;/p&gt;

&lt;p&gt;PDF file- 142K, The RET-PTC-RAS-BRAF cascade triggers CD44 cleavage&lt;/p&gt;

&lt;p&gt;PDF file - 102K, PKA and MEK inhibition blunts CD44-ICD mediated CREB phosphorylation&lt;/p&gt;

&lt;p&gt;PDF file - 141K, RET-PTC triggers CD44-ICD and CD44-CTF production&lt;/p&gt;

&lt;p&gt;PDF file- 142K, The RET-PTC-RAS-BRAF cascade triggers CD44 cleavage&lt;/p&gt;

&lt;p&gt;PDF file - 175K, CD44-ICD-CREB complex in human PTC cell lines&lt;/p&gt;

&lt;p&gt;PDF file - 102K, PKA and MEK inhibition blunts CD44-ICD mediated CREB phosphorylation&lt;/p&gt;

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&lt;p&gt;PDF file - 141K, RET-PTC triggers CD44-ICD and CD44-CTF production&lt;/p&gt;

&lt;p&gt;PDF file - 78K, CD44-ICD expression in human thyroid cancer cell lines&lt;/p&gt;

&lt;p&gt;PDF file - 106K, Supplemental table 1. Antibodies used in the study; Supplemental table 2. Genes differentially expressed in MM1.S cells exposed to IGF-1 (200 ng/mL) and bortezomib (10 nM). The list indicate genes with at least a 2-fold difference in expression levels (Agilent pangenomic array)&lt;/p&gt;

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&lt;p&gt;PDF file - 464K, Results shown are representative of three separate experiments (S1C, S2, S3A) and mean values from 3 independent experiments +/- SD (S1A-B, S4)&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Multiple myeloma is a clonal plasma cell disorder in which growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type I (IGF-I). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We analyzed the impact of IGF-I combined with proteasome inhibitors on multiple myeloma cell lines &lt;i&gt;in vivo&lt;/i&gt; and &lt;i&gt;in vitro&lt;/i&gt; as well as on fresh human myeloma cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Our study shows that IGF-I enhances the cytotoxic effect of proteasome inhibitors against myeloma cells. The effect of bortezomib on the content of proapoptotic proteins such as Bax, Bad, Bak, and BimS and antiapoptotic proteins such as Bcl-2, Bcl-XL, XIAP, Bfl-1, and survivin was enhanced by IGF-I. The addition of IGF-I to bortezomib had a minor effect on NF-κB signaling in MM.1S cells while strongly enhancing reticulum stress. This resulted in an unfolded protein response (UPR), which was required for the potentiating effect of IGF-I on bortezomib cytotoxicity as shown by siRNA-mediated inhibition of GADD153 expression.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; These results suggest that the high baseline level of protein synthesis in myeloma can be exploited therapeutically by combining proteasome inhibitors with IGF-I, which possesses a “priming” effect on myeloma cells for this family of compounds. &lt;i&gt;Clin Cancer Res; 19(13); 3556–66. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Multiple myeloma is a clonal plasma cell disorder in which growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type I (IGF-I). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We analyzed the impact of IGF-I combined with proteasome inhibitors on multiple myeloma cell lines &lt;i&gt;in vivo&lt;/i&gt; and &lt;i&gt;in vitro&lt;/i&gt; as well as on fresh human myeloma cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Our study shows that IGF-I enhances the cytotoxic effect of proteasome inhibitors against myeloma cells. The effect of bortezomib on the content of proapoptotic proteins such as Bax, Bad, Bak, and BimS and antiapoptotic proteins such as Bcl-2, Bcl-XL, XIAP, Bfl-1, and survivin was enhanced by IGF-I. The addition of IGF-I to bortezomib had a minor effect on NF-κB signaling in MM.1S cells while strongly enhancing reticulum stress. This resulted in an unfolded protein response (UPR), which was required for the potentiating effect of IGF-I on bortezomib cytotoxicity as shown by siRNA-mediated inhibition of GADD153 expression.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; These results suggest that the high baseline level of protein synthesis in myeloma can be exploited therapeutically by combining proteasome inhibitors with IGF-I, which possesses a “priming” effect on myeloma cells for this family of compounds. &lt;i&gt;Clin Cancer Res; 19(13); 3556–66. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;PDF file - 464K, Results shown are representative of three separate experiments (S1C, S2, S3A) and mean values from 3 independent experiments +/- SD (S1A-B, S4)&lt;/p&gt;

&lt;p&gt;PDF file - 106K, Supplemental table 1. Antibodies used in the study; Supplemental table 2. Genes differentially expressed in MM1.S cells exposed to IGF-1 (200 ng/mL) and bortezomib (10 nM). The list indicate genes with at least a 2-fold difference in expression levels (Agilent pangenomic array)&lt;/p&gt;

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&lt;div&gt;Abstract&lt;p&gt;CD44 is a marker of cancer stem-like cells and epithelial–mesenchymal transition that is overexpressed in many cancer types, including thyroid carcinoma. At extracellular and intramembranous domains, CD44 undergoes sequential metalloprotease- and γ-secretase–mediated proteolytic cleavage, releasing the intracellular protein fragment CD44-ICD, which translocates to the nucleus and activates gene transcription. Here, we show that CD44-ICD binds to the transcription factor CREB, increasing S133 phosphorylation and CREB-mediated gene transcription. CD44-ICD enhanced CREB recruitment to the cyclin D1 promoter, promoting cyclin D1 transcription and cell proliferation. Thyroid carcinoma cells harboring activated RET/PTC, RAS, or BRAF oncogenes exhibited CD44 cleavage and CD44-ICD accumulation. Chemical blockade of RET/PTC, BRAF, metalloprotease, or γ-secretase were each sufficient to blunt CD44 processing. Furthermore, thyroid cancer cell proliferation was obstructed by RNA interference–mediated knockdown of CD44 or inhibition of γ-secretase and adoptive CD44-ICD overexpression rescued cell proliferation. Together, these findings reveal a CD44-CREB signaling pathway that is needed to sustain cancer cell proliferation, potentially offering new molecular targets for therapeutic intervention in thyroid carcinoma. &lt;i&gt;Cancer Res; 72(6); 1449–58. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;CD44 is a marker of cancer stem-like cells and epithelial–mesenchymal transition that is overexpressed in many cancer types, including thyroid carcinoma. At extracellular and intramembranous domains, CD44 undergoes sequential metalloprotease- and γ-secretase–mediated proteolytic cleavage, releasing the intracellular protein fragment CD44-ICD, which translocates to the nucleus and activates gene transcription. Here, we show that CD44-ICD binds to the transcription factor CREB, increasing S133 phosphorylation and CREB-mediated gene transcription. CD44-ICD enhanced CREB recruitment to the cyclin D1 promoter, promoting cyclin D1 transcription and cell proliferation. Thyroid carcinoma cells harboring activated RET/PTC, RAS, or BRAF oncogenes exhibited CD44 cleavage and CD44-ICD accumulation. Chemical blockade of RET/PTC, BRAF, metalloprotease, or γ-secretase were each sufficient to blunt CD44 processing. Furthermore, thyroid cancer cell proliferation was obstructed by RNA interference–mediated knockdown of CD44 or inhibition of γ-secretase and adoptive CD44-ICD overexpression rescued cell proliferation. Together, these findings reveal a CD44-CREB signaling pathway that is needed to sustain cancer cell proliferation, potentially offering new molecular targets for therapeutic intervention in thyroid carcinoma. &lt;i&gt;Cancer Res; 72(6); 1449–58. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Within secondary lymphoid organs, B and T lymphocytes are not randomly distributed but they specifically inhabit highly organized regions that contain areas where B and T cells are largely segregated and other areas where different populations of cells can interact and differentiate (Thorbecke et al. 1962). Specifically, in lymph nodes and tonsils, the majority of B cells localize to follicles, whereas most T cells are found in the paracortex (Nieuwenhuis and Ford 1976). Following initial entrance into the interfollicular areas of secondary lymphoid tissues, it is not known what drives B cells to localize to follicles; however, the formation of follicles is an antigen (Ag)-driven process that requires the presence and participation of T lymphocytes.

Abstract Abstract 3987 Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma and has been suggested to induce apoptotsis by reducing NF-κB signalling. Other cytotoxic mechanisms have been suggested, including increased reticulum stress leading to an unfolded protein response. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib on human plasma cell lines in vitro and in vivo and on fresh human myeloma cells ex vivo. Using an MTT assay, we found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro against the LP1, RPMI8226, U266 and MM1.S lines, at a concentration of IGF-1 of 100 ng/mL. This potentiating effect was confirmed on MM1.S cells using a flow cytometric analysis of annexin V staining, and showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 was also found to enhance the cytotoxic activity of other proteasome inhibitors against MM1.S cells, including MG115, MG132, PSI and epoximicin. In vivo studies were performed in SCID mice bearing MM1.S xenografts. Mice received weekly administrations of bortezomib (0.5 mg/kg, i.p.) with or without recombinant IGF-1 (0.03 mg/kg, i.p.). The co-administration of IGF-1 with bortezomib significantly delayed tumor growth in comparison to that observed in mice treated with bortezomib alone. Fresh human myeloma cells exposed to bortezomib ex vivo displayed a larger annexin V positive fraction when they were co-incubated with IGF-1 then when they were exposed to bortezomib alone. This effect, which could be observed in subpopulations of CD45 hi and CD45 lo cells, could be reversed by an antibody directed against IGF-1R. Thus in each of these situations, IGF-1 increased the sensitivity of multiple myeloma cells to the cytotoxic effect of bortezomib. Analysis of pro- and anti-apoptotic proteins in MM1.S cells by immunoblotting showed that the addition of IGF-1 to bortezomib significantly enhanced the content o Bax, Bad and Bak and significantly reduced the content of Bcl2, BclX-L and Bfl-1. Exploration the NFkB pathway showed that exposure to IGF-1 and bortezomib induced a reduction of IkBalpha, an increase in phosphor-IKBalpha as well as a decrease in NFkB p65. Other observations made with the IGF-1/bortezomib combination include an increase in the content of cleaved caspase 3 and in P21 protein. Cell cycle distributions of cells exposed to bortezomib alone or the IGF-1/bortezomib combination were similar. Preliminary data showed an increased content of CHOP protein, suggesting that the IGF-1/bortezomib combination might enhance reticulum stress in MM1.S cells, thus leading to an Unfolded Protein Response (UPR) and to cell death. These results suggest that IGF-1 sensitizes myeloma cells to proteasome inhibitors by contributing to the enhancement of the reticulum stress. Overall these results suggest that exposure of myeloma cells to one of their key growth factors, IGF-1, significantly enhanced their sensitivity to bortezomib as well as to other proteasome inhibitors. This phenomenon appears to involve several pathways and may be dependent on the high baseline level of reticulum stress present in myeloma cells. Disclosures: No relevant conflicts of interest to declare.

Abstract Deregulation of nucleotide metabolism can lead to biological disturbances such as genetic instability, energetic homeostasis deregulation and pro-proliferative signaling, that are among the “Hallmarks of Cancer” described by Hanahan and Weinberg. Thus, this complex process has become in hotspot in cancer research. To fully apprehend to what extent nucleotide metabolism can be targeted for new anti-cancer therapies, the involved molecular actors and their function in cancer cell biology need to be better understood. Purine metabolism involves various intracellular and extracellular enzymes including cN-II (cytosolic nucleotidase-II) and CD73 that are two 5'-nucleotidases respectively able to dephosphorylate intracellular and extracellular nucleoside monophosphates into corresponding nucleosides. Considering nucleotide/nucleoside trafficking and their roles in cell biology, it is a possible that cN-II and CD73 are involved together in processes that increase cancer cells aggressiveness. We abolished cN-II and/or CD73 expressions in two human carcinoma cell lines (MDA-MB-231 and NCI-H292), using the CRISPR/Cas9 technique, and evaluated the impact of cN-II and CD73 on cell migration, under an extracellular nucleotide stress (by performing wound healing assays on the Incucyte device). The obtained results showed that cN-II alone or together with CD73, was able to modify cell migration, in absence and in presence of an extracellular nucleotide stress. Indeed, cN-II deficiency was associated with accelerated cell migration, in control conditions. Moreover, exposure to adenosine decreased migration for all the models, with a stronger effect on cN-II deficient cells. We further investigated on the expression or activity of migration regulators, and found that 5'-nucleotidases deficiency was associated with altered transcriptional expression of gelatinases (MMP-2 and MMP9) and their negative regulator (TIMP2). The study of signaling pathways that are known to be related to cell migration are currently studied. These in vitro observations justify upcoming in vivo studies that aim to correlate these results with metastasis occurrence after xenografts in immunodeficient mice. For the first time, our results define cN-II as involved in cell migration, confirming that this 5'-nucleotidase could represent an interesting target to reduce pro metastatic behaviors in cancer cells. Nevertheless, it remains to be defined whether the relation between 5'-nucleotidases and cell migration involves their catalytic activity (and thus nucleotide pools balance) or if it is due to the physical interaction with complexes that are already known to regulate this process. Citation Format: Octavia Cadassou, Muhammad Zawwad Raza, Emeline Cros-Perrial, Célia Armanet, Laura Gudefin, Kamel Chettab, Serge Manié, Charles Dumontet, Lars P. Jordheim. Impact of cN-II and CD73 inhibition on cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3164.

Among the cellular adhesion molecules, the integrin family, more particularly the VLA (Very late antigen) integrins, is currently the subject of numerous investigations in pathology. These integrins are involved in cell-cell contact or cell-matrix adhesions. During neoplastic diseases, cellular expression of integrins changes and a study of the modifications could allow a new etiopathogenic approach carcinogenesis and metastatic phenomena. New prognostic factors may be defined in tumor pathology. We describe the general structure of integrins and the mechanisms of their binding with matricial ligands and with cytoskeleton. The expression of VLA integrins and the alpha6beta4 heterodimer on normal and neoplastic human tissues is then described. Finally, we describe the involvement of these proteins in tumor progression and tissue invasion.

A convenient and sensitive sequential sandwich colorimetric ELISA test was established for quantitating IL-6 in culture supernatants or in serum. Immunopurified HRP-labelled rabbit Fab' fragment was used as the tracer and IgG-coated microtiter plate as the capture antibody. The limit of detection was as low as 10 attomoles of analyte (2.5 pg/ml). Unglycosylated recombinant IL-6 and the natural glycosylated cytokine were recognized equally. In addition, IL-6 measurements were unaffected by the presence of various cytokines and assay sensitivity was only slightly reduced in the presence of undiluted serum samples. The technique was applied to the study of in vitro IL-6 production from activated monocytes and to the in vivo determination of IL-6 in various pathological states.

The tetraspans associate with a large number of surface molecules, including a subset of β1 integrins and, indirectly through CD19, with the complement receptor CD21. To further characterize the tetraspan complexes we have raised and selected monoclonal antibodies (mAb) for their ability to immunoprecipitate a molecule associated with CD9. A unique mAb was identified which recognizes the complement regulator CD46 (membrane cofactor protein). CD46 associated in part with several tetranspans and with all β1 integrins that were tested (CD29 / CD49a, CD29 / CD49b, CD29 / CD49c, CD29 / CD49e, CD29 / CD49f) but not with β4 integrins. These data, together with cross-linking experiments showing the existence in living cells of CD46 / integrin complexes, suggest that CD46 associates directly with β1 integrins and indirectly with tetraspans. CD46 also acts as a receptor for measles virus; however, mAb to various integrins and tetraspans did not modify the virus fusion entry step.

L'interleukine-1 (il-1), cytokine pro-inflammatoire et multifonctionnelle, joue un role important comme co-facteur dans le processus d'activation lymphocytaire. Cependant, les mecanismes qui sous-tendent son induction lors de la presentation antigenique ainsi que les voies de signalisation intracellulaire qui regulent sa synthese sont pour une grande part mal definis. Nous avons, d'une part, etudie l'induction d'il-1 a partir d'un modele d'interaction cellulaire entre des lymphocytes t (lignee jurkat) et des monocytes (lignee thp-1). Les resultats obtenus montrent que la cellule jurkat partiellement activee par la voie cd3/tcr, stimule la production d'il-1 a partir des cellules thp-1. Cette induction, strictement dependante d'un contact cellulaire, implique de maniere differente les molecules de surface lfa-1 et cd69, suggerant un nouveau role pour cette derniere dans la stimulation de la cellule presentatrice de l'antigene. D'autre part, nous avons fait l'observation originale que les drogues destructurant les microtubules, stimulent selectivement l'expression des genes de l'il-1 et il-1 et la production de leurs proteines correspondantes, l'il-6 et le tnf- n'etant pas, dans ces conditions, stimules. A l'inverse, les cytochalasines qui desorganisent les filaments d'actine, sont depourvues d'effets sur la production de l'il-1. Cette stimulation semble necessiter une depolymerisation du reseau microtubulaire et implique au moins deux voies de signalisation. La premiere resulte d'une augmentation des taux d'ampc intracellulaire et d'une activation de la proteine kinase a. La seconde impliquerait la stimulation de proteines tyrosine kinases. Ce travail suggere en outre, que la reorganisation du cytosquelette contribue aux mecanismes de transduction du signal