Cancer cell-derived long pentraxin 3 (PTX3) promotes melanoma migration through a toll-like receptor 4 (TLR4)/NF-κB signaling pathway.

Cutaneous melanoma is one of the most aggressive cancers characterized by a high plasticity, a propensity for metastasis, and drug resistance. Melanomas are composed of phenotypically diverse subpopulations of tumor cells with heterogeneous molecular profiles that reflect intrinsic invasive abilities. In an attempt to identify novel factors of the melanoma invasive cell state, we previously investigated the nature of the invasive secretome by using a comparative proteomic approach. Here, we have extended this analysis to show that PTX3, an acute phase inflammatory glycoprotein, is one such factor secreted by invasive melanoma to promote tumor cell invasiveness. Elevated PTX3 production was observed in the population of MITFlow invasive cells but not in the population of MITFhigh differentiated melanoma cells. Consistently, MITF knockdown increased PTX3 expression in MITFhigh proliferative and poorly invasive cells. High levels of PTX3 were found in tissues and blood of metastatic melanoma patients, and in BRAF inhibitor-resistant melanoma cells displaying a mesenchymal invasive MITFlow phenotype. Genetic silencing of PTX3 in invasive melanoma cells dramatically impaired migration and invasion in vitro and in experimental lung extravasation assay in xenografted mice. In contrast, addition of melanoma-derived or recombinant PTX3, or expression of PTX3 enhanced motility of low migratory cells. Mechanistically, autocrine production of PTX3 by melanoma cells triggered an IKK/NFκB signaling pathway that promotes migration, invasion, and expression of the EMT factor TWIST1. Finally, we found that TLR4 and MYD88 knockdown inhibited PTX3-induced melanoma cell migration, suggesting that PTX3 functions through a TLR4-dependent pathway. Our work reveals that tumor-derived PTX3 contributes to melanoma cell invasion via targetable inflammation-related pathways. In addition to providing new insights into the biology of melanoma invasive behavior, this study underscores the notion that secreted PTX3 represents a potential biomarker and therapeutic target in a subpopulation of MITFlow invasive and/or refractory melanoma.

Hypoxia and MITF control metastatic behaviour in mouse and human melanoma cells.

Melanomas are very aggressive neoplasms with notorious resistance to therapeutics. It was recently proposed that the remarkable phenotypic plasticity of melanoma cells allows for the rapid development of both resistance to chemotherapeutic drugs and invasive properties. Indeed, the capacity of melanoma cells to form distant metastases is the main cause of mortality in melanoma patients. Therefore, the identification of the mechanism controlling melanoma phenotype is of paramount importance. In the present report, we show that deletion of microphthalmia-associated transcription factor (MITF), the master gene in melanocyte differentiation, is sufficient to increase the metastatic potential of mouse and human melanoma cells. MITF silencing also increases fibronectin and Snail, two mesenchymal markers that might explain the increased invasiveness in vitro and in vivo. Furthermore, ablation of this population by Forskolin-induced differentiation or MITF-forced expression significantly decreases tumour and metastasis formation, suggesting that eradication of low-MITF cells might improve melanoma treatment. Moreover, we demonstrate that a hypoxic microenvironment decreases MITF expression through an indirect, hypoxia-inducible factor 1 (HIF1)α-dependant transcriptional mechanism, and increases the tumourigenic and metastatic properties of melanoma cells. We identified Bhlhb2, a new factor in melanoma biology, as the mediator of hypoxia/HIF1α inhibitory effect on MITF expression. Our results reveal a hypoxia-HIF1α-BHLHB2-MITF cascade controlling the phenotypic plasticity in melanoma cells and favouring metastasis development. Targeting this pathway might be helpful in the design of new anti-melanoma therapies.

Metformin inhibits melanoma development through autophagy and apoptosis mechanisms.

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.

SPARC functions as an anti-stress factor by inactivating p53 through Akt-mediated MDM2 phosphorylation to promote melanoma cell survival.

Aberrant expression of Secreted Protein Acidic and Rich in Cysteine (SPARC)/osteonectin has been associated with an invasive tumor cell phenotype and poor outcome in human melanomas. Although it is known that SPARC controls melanoma tumorigenesis, the precise role of SPARC in melanoma cell survival is still unclear. Here, we show that SPARC has a cell-autonomous survival activity, which requires Akt-dependent regulation of p53. Suppression of SPARC by RNA interference in several human melanoma cells and xenografted A375 tumors triggers apoptotic cell death through the mitochondrial intrinsic pathway and activation of caspase-3. Cell death induced by depletion of SPARC is dependent on p53 and induction of Bax, and results in the generation of ROS. Stabilization of p53 in SPARC-depleted cells is associated with a decrease in Akt-mediated activating phosphorylation of MDM2. Inhibition of Akt signaling pathway is important for the observed changes as overexpression of constitutively active Akt protects cells against apoptosis induced by SPARC depletion. Conversely, increased expression of SPARC stimulates Akt and MDM2 phosphorylation, thus facilitating p53 degradation. Finally, we show that overexpression of SPARC renders cells more resistant to the p53-mediated cytotoxic effects of the DNA-damaging drug actinomycin-D. Our study indicates that SPARC functions through activation of Akt and MDM2 to limit p53 levels and that acquired expression of SPARC during melanoma development would confer survival advantages through suppression of p53-dependent apoptotic pathways.

Ciglitazone negatively regulates CXCL1 signaling through MITF to suppress melanoma growth.

We have previously demonstrated that the thiazolidinedione ciglitazone inhibited, independently of PPARγ activation, melanoma cell growth. Further investigations now show that ciglitazone effects are mediated through the regulation of secreted factors. Q-PCR screening of several genes involved in melanoma biology reveals that ciglitazone inhibits expression of the CXCL1 chemokine gene. CXCL1 is overexpressed in melanoma and contributes to tumorigenicity. We show that ciglitazone induces a diminution of CXCL1 level in different human melanoma cell lines. This effect is mediated by the downregulation of microphthalmia-associated transcription factor, MITF, the master gene in melanocyte differentiation and involved in melanoma development. Further, recombinant CXCL1 protein is sufficient to abrogate thiazolidinedione effects such as apoptosis induction, whereas extinction of the CXCL1 pathway mimics phenotypic changes observed in response to ciglitazone. Finally, inhibition of human melanoma tumor development in nude mice treated with ciglitazone is associated with a strong decrease in MITF and CXCL1 levels. Our results show that anti-melanoma effects of thiazolidinediones involve an inhibition of the MITF/CXCL1 axis and highlight the key role of this specific pathway in melanoma malignancy.

The tyrosine kinase Syk regulates the survival of chronic lymphocytic leukemia B cells through PKCdelta and proteasome-dependent regulation of Mcl-1 expression.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by accumulation of mature monoclonal CD5+ B cells. The disease results mainly from a failure of cells to undergo apoptosis, a process largely influenced by the existence of constitutively activated components of B-cell receptor signaling and the deregulated expression of anti-apoptotic molecules. Recent evidence pointing to a critical role of spleen tyrosine kinase (Syk) in ligand-independent BCR signaling prompted us to examine its role in primary B-CLL cell survival. We demonstrate that pharmacological inhibition of constitutive Syk activity and silencing by siRNA led to a dramatic decrease of cell viability in CLL samples (n=44), regardless of clinical and biological status and induced typical apoptotic cell death with mitochondrial failure followed by caspase 3-dependent cell death. We also provide functional and biochemical evidence that Syk regulated B-CLL cell survival through a novel pathway involving PKCdelta and a proteasome-dependent regulation of the anti-apoptotic protein Mcl-1. Together, our observations are consistent with a model wherein PKCdelta downstream of Syk stabilizes Mcl-1 through inhibitory phosphorylation of GSK3 by Akt. We conclude that Syk constitutes a key regulator of B-CLL cell survival, emphasizing the clinical utility of Syk inhibition in hematopoietic malignancies.

PI3K mediates protection against TRAIL-induced apoptosis in primary human melanocytes.

Melanocytes are cells of the epidermis that synthesize melanin, which is responsible for skin pigmentation. Transformation of melanocytes leads to melanoma, a highly aggressive neoplasm, which displays resistance to apoptosis. In this report, we demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), which was thought to kill only transformed cells, promotes very efficiently apoptosis of primary human melanocytes, leading to activation of caspases 8, 9 and 3, and the cleavage of vital proteins. Further, we show that stem cell factor (SCF), a physiologic melanocyte growth factor that activates both the phosphatidyl-inositol-3 kinase (PI3K) and the extracellular regulated kinase (ERK) pathways, strongly protects melanocytes from TRAIL and staurosporine killing. Interestingly, inhibition of PI3K or its downstream target AKT completely blocks the antiapoptotic effect of SCF, while inhibition of ERK has only a moderate effect. Our data indicate that protection evoked by SCF/PI3K/AKT cascade is not mediated by an increase in the intracellular level of FLIP. Further, only a sustained PI3K activity can protect melanocytes from apoptosis, thereby indicating that the PI3K/AKT pathway plays a pivotal role in melanocyte survival. The results gathered in this report bring new information on the molecular mechanisms involved in primary melanocyte apoptosis and survival that would help to better understand the process by which melanomas acquire their resistance to apoptosis.

Surfing the insulin signaling web.

The diverse biological actions of insulin and insulin-like growth factor I (IGF-I) are initiated by binding of the polypeptides to their respective cell surface tyrosine kinase receptors. These activated receptors phosphorylate a series of endogenous substrates on tyrosine, amongst which the insulin receptor substrate (IRS) proteins are the best characterized. Their phosphotyrosine-containing motifs become binding sites for Src homology 2 (SH2) domains on proteins such as SH2 domain-containing protein-tyrosine-phosphatase (SHP)-2/Syp, growth factor receptor bound-2 protein, (Grb-2), and phosphatidyl inositol 3 kinase (PI3 kinase), which participate in activation of specific signaling cascades. However, the IRS molecules are not only platforms for signaling molecules, they also orchestrate the generation of signal specificity, integration of signals induced by several extracellular stimuli, and signal termination and modulation. An extensive review is beyond the scope of the present article, which will be centered on our own contribution and reflect our biases.

The matricellular protein SPARC/osteonectin as a newly identified factor up-regulated in obesity.

Alterations in the expression level of genes may contribute to the development and pathophysiology of obesity. To find genes differentially expressed in adipose tissue during obesity, we performed suppression subtractive hybridization on epididymal fat mRNA from goldthioglucose (GTG) obese mice and from their lean littermates. We identified the secreted protein acidic and rich in cysteine (SPARC), a protein that mediates cell-matrix interactions and plays a role in modulation of cell adhesion, differentiation, and angiogenesis. SPARC mRNA expression in adipose tissue was markedly increased (between 3- and 6-fold) in three different models of obesity, i.e. GTG mice, ob/ob mice, and AKR mice, after 6 weeks of a high fat diet. Immunoblotting of adipocyte extracts revealed a similar increase in protein level. Using a SPARC-specific ELISA, we demonstrated that SPARC is secreted by isolated adipocytes. We found that insulin administration to mice increased SPARC mRNA in the adipose tissue. Food deprivation had no effect on SPARC expression, but after high fat refeeding SPARC mRNA levels were significantly increased. Our results reveal both hormonal and nutritional regulation of SPARC expression in the adipocyte, and importantly, its alteration in obesity. Finally, we show that purified SPARC increased mRNA levels of plasminogen activator inhibitor 1 (PAI-1) in cultured rat adipose tissue suggesting that elevated adipocyte expression of SPARC might contribute to the abnormal expression of PAI-1 observed in obesity. We propose that SPARC is a newly identified autocrine/paracrine factor that could affect key functions in adipose tissue physiology and pathology.

Vav2 activates c-fos serum response element and CD69 expression but negatively regulates nuclear factor of activated T cells and interleukin-2 gene activation in T lymphocyte.

Vav1 and Vav2 are members of the Dbl family of guanine nucleotide exchange factors for the Rho family of small GTPases. Although the role of Vav1 during lymphocyte development and activation is well characterized, the function of Vav2 is still unclear. In this study, we compared the signaling pathways regulated by Vav1 and Vav2 following engagement of the T cell receptor (TCR). We show that Vav2 is tyrosine-phosphorylated upon TCR stimulation and by co-expressed Src and Syk family kinases. Using glutathione S-transferase fusion proteins, we observed that the Src homology 2 domain of Vav2 binds tyrosine-phosphorylated proteins from TCR-stimulated Jurkat T cell lysates, including c-Cbl and SLP-76. Like Vav1, Vav2 cooperated with TCR stimulation to increase extracellular signal-regulated kinase activation and to promote c-fos serum response element transcriptional activity. Moreover, both proteins displayed a similar action in increasing the expression of the early activation marker CD69 in Jurkat T cells. However, in contrast to Vav1, Vav2 dramatically suppressed TCR signals leading to nuclear factor of activated T cells (NF-AT)-dependent transcription and induction of the interleukin-2 promoter. Vav2 appears to act upstream of the phosphatase calcineurin because a constitutively active form of calcineurin rescued the effect of Vav2 by restoring TCR-induced NF-AT activation. Interestingly, the Dbl homology and Src homology 2 domains of Vav2 were necessary for its inhibitory effect on NF-AT activation and for induction of serum response element transcriptional activity. Taken together, our results indicate that Vav1 and Vav2 exert overlapping but nonidentical functions in T cells. The negative regulatory pathway elicited by Vav2 might play an important role in regulating lymphocyte activation processes.

Stat 5B, activated by insulin in a Jak-independent fashion, plays a role in glucokinase gene transcription.

Stat proteins are SH2 domain-containing transcription factors that are activated by various cytokines and growth factors. In a previous work, we have identified Stat 5B as a substrate of the insulin receptor based on yeast two-hybrid and mammalian cell transfection studies. In the present study, we have approached the biological relevance of the interaction between the insulin receptor and the transcription factor Stat 5B. Firstly, we show that both insulin and insulin-like growth factor I lead to tyrosine phosphorylation of Stat 5B, and this promotes binding of the transcription factor to the beta-casein promoter containing a Stat 5 binding site. Further, we demonstrate that insulin stimulates the transcriptional activity of Stat 5B. Activation of Stat 5B by insulin appears to be Jak2-independent, whereas Jak2 is required for GH-induced Stat 5B activation. Hence the pathway by which Stat 5B is activated by insulin is different from that used by GH. In addition, by using Jak1- and Tyk2-deficient cells we exclude the involvement of both Jak1 and Tyk2 in Stat 5B activation by insulin. Taken together, our results strengthen the notion that insulin receptor can directly activate Stat 5B. More importantly, we have identified a Stat 5 binding site in the human hepatic glucokinase promoter, and we show that insulin leads to a Stat 5B-dependent increase in transcription of a reporter gene carrying this promoter. These observations favor the idea that Stat 5B plays a role in mediating the expression of the glucokinase gene induced by insulin. As a whole, our results provide evidence for the occurrence of a newly identified circuit in insulin signaling in which the cell surface receptor is directly linked to nuclear events through a transcription factor. Further, we have revealed an insulin target gene whose expression is, at least in part, dependent on Stat 5B activation and/or binding.

Two distinct mutations of the RET receptor causing Hirschsprung's disease impair the binding of signalling effectors to a multifunctional docking site.

The RET gene codes for a transmembrane tyrosine kinase which is a subunit of a multimeric complex that acts as a receptor for four structurally related molecules: the glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin and persephin. Germline mutations of RET cause a dominantly inherited dysgenesis of the enteric nervous system known as Hirschsprung's disease (HSCR; aganglionosis megacolon). The majority of HSCR mutations results either in a reduction of dosage of the RET protein or in the loss of RET function. Two novel distinct mutations of RET that led either to the deletion of codon 1059 (denoted Delta1059) or to the substitution of a Pro for Leu1061 have been identified in five HSCR families. In one large pedigree, two children born from asymptomatic consanguineous parents presented a severe form of HSCR and were found to carry the mutation at codon 1061 in the homozygous state. A tyrosine residue at position 1062 is an intracytoplasmic docking site that enables RET to recruit several signalling molecules, including the Shc adaptor protein. We now report that both HSCR mutations impair the fixation of Shc to RET and consequently prevent its phosphorylation. In addition, quantitative analysis in PC12 cells reveals that mutation Delta1059 inactivates the ability of RET to transduce a downstream signal whereas mutation L1061P only partially inhibits the signalling of RET. Finally, we provide evidence that these effects are partly mediated via the disruption of the RET/Shc interaction. Collectively, these results demonstrate that HSCR can be ascribed to mutations of RET which interfere with the binding of transduction effectors, such as Shc, and further provide a biochemical explanation for the phenotype of patients carrying a homozygous mutation at codon 1061. Finally, these data indicate that Y1062 is a multifunctional docking site that confers to RET the capacity to engage downstream signalling pathways which exert a crucial role during enteric neurogenesis.

C-terminal Src kinase associates with ligand-stimulated insulin-like growth factor-I receptor.

Increased expression of the insulin-like growth factor-I receptor (IGF-IR) protein-tyrosine kinase occurs in several kinds of cancer and induces neoplastic transformation in fibroblast cell lines. The transformed phenotype can be reversed by interfering with the function of the IGF-IR. The IGF-IR is required for transformation by a number of viral and cellular oncoproteins, including SV40 large T antigen, Ras, Raf, and Src. The IGF-IR is a substrate for Src in vitro and is phosphorylated in v-Src-transformed cells. We observed that the IGF-IR and IR associated with the C-terminal Src kinase (CSK) following ligand stimulation. We found that the SH2 domain of CSK binds to the tyrosine-phosphorylated form of IGF-IR and IR. We determined the tyrosine residues in the IGF-IR and in the IR responsible for this interaction. We also observed that fibroblasts stimulated with IGF-I or insulin showed a rapid and transient decrease in c-Src tyrosine kinase activity. The results suggest that c-Src and CSK are involved in IGF-IR and IR signaling and that the interaction of CSK with the IGF-IR may play a role in the decrease in c-Src activity following IGF-I stimulation.

Adaptor function for the Syk kinases-interacting protein 3BP2 in IL-2 gene activation.

Syk-family tyrosine kinases are essential for lymphocyte development and activation. Using a yeast two-hybrid screen to identify Syk kinases-interacting proteins (SKIPs), we isolated 3BP2, an Abl SH3-interacting protein of unknown function. 3BP2 was selectively expressed in hematopoietic/lymphoid tissues and bound via its SH2 domain activated Syk-family kinases in mammalian cells, including in antigen receptor-stimulated T cells. In addition to Zap-70, the 3BP2 SH2 domain associated in vitro with LAT, Grb2, PLCgamma1, and Cbl from activated T cell lysates. Transient 3BP2 overexpression induced transcriptional activation of the IL-2 promoter and its NFAT or AP-1 elements. This activity was dependent on the SH2 and pleckstrin-homology domains of 3BP2, and required functional Syk kinases, Ras, and calcineurin. Thus, 3BP2 is an important adaptor that may couple activated Zap-70/Syk to a LAT-containing signaling complex involved in TCR-mediated gene transcription.

Determination of Gab1 (Grb2-associated binder-1) interaction with insulin receptor-signaling molecules.

The newly identified insulin receptor (IR) substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] is rapidly phosphorylated on several tyrosine residues by the activated IR. Phosphorylated Gab1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins. These include the regulatory subunit p85 of phosphatidylinositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this report, using a modified version of the yeast two-hybrid system, we localized which Gab1 phospho-tyrosine residues are required for its interaction with phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 589 are participating in this process. Concerning Gab1/SHP-2 interaction, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Finally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/ Gab1 interaction and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.

Evidence for a direct interaction between insulin receptor substrate-1 and Shc.

Insulin receptor substrate-1 (IRS-1) and Shc are two proteins implicated in intracellular signal transduction. They are activated by an increasing number of extracellular signals, mediated by receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors. In this study we demonstrate that Shc interacts directly with IRS-1, using the yeast two-hybrid system and an in vitro interaction assay. Deletion analysis of the proteins to map the domains implicated in this interaction shows that the phosphotyrosine binding domain of Shc binds to the region of IRS-1 comprising amino acids 583-661. An in vitro association assay, performed with or without activation of tyrosine kinases, gives evidence that tyrosine phosphorylation of IRS-1 and Shc drastically improves the interaction. Site-directed mutagenesis on IRS-1 583-693 shows that the asparagine, but not the tyrosine residue of the N625GDY628motif domain, is implicated in the IRS-1-Shc-phosphotyrosine binding interaction. Mutation of another tyrosine residue, Tyr608, also induced a 40% decrease in the interaction. This study, describing a phosphotyrosine-dependent interaction between IRS-1 and Shc, suggests that this association might be important in signal transduction.

Identification of Stat 5B as a substrate of the insulin receptor.

We have screened a human placenta library using the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Stat 5B and the receptor requires a functional insulin-receptor kinase, Tyr960 of insulin receptor is implicated in the interaction with Stat 5B, whereas asparagine and proline forming the NPEY960-motif are not, and Stat 5B mutated at Thr684, a potential phosphorylation site of mitogen-activated protein kinase, loses its ability to interact with the insulin receptor. Further, we found that insulin promotes rapid tyrosine phosphorylation of endogenous Stat 5B in 293 EBNA cells overexpressing insulin receptor and in NHIR cells. Taken together, our findings suggest that Stat 5B corresponds to a substrate for the insulin-receptor kinase, and this widens the repertoire of insulin-signaling pathways.

Functional and physical interactions of Syk family kinases with the Vav proto-oncogene product.

Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.

Interaction of SH2-containing protein tyrosine phosphatase 2 with the insulin receptor and the insulin-like growth factor-I receptor: studies of the domains involved using the yeast two-hybrid system.

Activated insulin and insulin-like growth factor-I receptors transmit downstream signals via the insulin receptor substrate (IRS-1 and IRS-2) and a series of proteins containing Src homology-2 (SH2) domains, including SH2-containing protein tyrosine phosphatase 2 (SHP-2). In the present study, we analyzed in the yeast two-hybrid system the interaction between both receptors and SHP-2. We found that a catalytically inactive SHP-2 is able to bind to tyrosine-phosphorylated IR beta-subunit and IGF-I R beta-subunit. However, with wild-type SHP-2, we were unable to detect an interaction with these receptors, which is likely to be due to dephosphorylation of the receptors by the phosphatase. Further, our results demonstrate that tyrosine 1322 of the IR, and the corresponding tyrosine 1316 of the IGF-I R are implicated in the interaction with the SHP-2 SH2 domain. At the level of SHP-2, it would appear that both SH2 domains of SHP-2 are necessary for optimal association with either receptor. Finally, using several insulin and IGF-I receptor mutants, we found that the kinase regulatory autophosphorylation sites play an important role in the interaction of these receptors with the SHP-2 SH2 domain. These sites are also necessary for the interaction with full-length IRS-1. We conclude that 1) the IR and IGF-I R directly interact with SHP-2; 2) the C-terminus autophosphorylation of these receptors sites are involved in this process; and 3) the receptors' kinase autophosphorylation sites are necessary for the interaction with SHP-2 and also with IRS-1.

Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591-786.

We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system. Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively. The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1. Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1. This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation.