Insulin receptor substrate 4 supports insulin- and interleukin 4-stimulated proliferation of hematopoietic cells.

Signaling from the activated insulin receptor is initiated by its tyrosine phosphorylation of the insulin receptor substrates (IRSs). The IRSs then act as docking/effector proteins for various signaling proteins containing src homology 2 domains. Four members of the IRS family, designated IRS-1 through IRS-4, have been identified. Although these IRSs show considerable structural homology, the extent to which they overlap in functions has not been explored in detail. The 32D hematopoietic cell line, which contains no detectable amounts of any IRS, provides a system in which to determine whether an IRS supports cell proliferation. Previous studies have shown that introduction of IRS-1 or -2 into 32D cells overexpressing the insulin and IL-4 receptors (32D-R cells) enables the cells to undergo mitogenesis in response to insulin and IL-4. In the present study, we have examined IRS-4, a member of the IRS family that we recently discovered, in this system. Expression of IRS-4 in 32D-R cells permitted the cells to undergo mitogenesis and continuous proliferation in response to insulin and IL-4. Immunoblotting of phosphotyrosine proteins showed that insulin and IL-4 elicited the tyrosine phosphorylation of IRS-4 in these cells. Thus, IRS-4, like IRS-1 and -2, can function in the signal transduction pathways linking insulin and IL-4 receptors to cell proliferation.     

Role of insulin receptor substrate-2 in interleukin-9-dependent proliferation

Interleukin-9 (IL-9) stimulation results in JAK, STAT and IRS1/2 phosphorylation. The role of IRS adaptor proteins in IL-9 signaling is not clear. We show that IL-9 induces IRS2 phosphorylation and association with phosphatidylinositol-3 kinase (PI 3-K) p85 subunit in TS1 cells and BaF/9R cells, which proliferate upon IL-9 stimulation. We observed a PI 3-K-dependent phosphorylation of protein kinase B (PKB) in TS1 cells, but not in BaF/9R, nor in other IL-9-dependent cell lines. Finally, 32D cells that were transfected with the IL-9 receptor but lack IRS expression survived in the presence of IL-9. Ectopic IRS1 expression allowed for IL-9-induced proliferation, in the absence of significant PKB phosphorylation.     

The Pleckstrin Homology and Phosphotyrosine Binding Domains of Insulin Receptor Substrate 1 Mediate Inhibition of Apoptosis by Insulin

Insulin and insulin-like growth factor 1 (IGF-1) evoke diverse biological effects through receptor-mediated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. We investigated the elements of IRS-1 signaling that inhibit apoptosis of interleukin 3 (IL-3)-deprived 32D myeloid progenitor cells. 32D cells have few insulin receptors and no IRS proteins; therefore, insulin failed to inhibit apoptosis during IL-3 withdrawal. Insulin stimulated mitogen-activated protein kinase in 32D cells expressing insulin receptors (32D^IR) but failed to activate the phosphatidylinositol 3 (PI 3)-kinase cascade or to inhibit apoptosis. By contrast, insulin stimulated the PI 3-kinase cascade, inhibited apoptosis, and promoted replication of 32D^IR cells expressing IRS-1. As expected, insulin did not stimulate PI 3-kinase in 32D^IR cells, which expressed a truncated IRS-1 protein lacking the tail of tyrosine phosphorylation sites. However, this truncated IRS-1 protein, which retained the NH₂-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, mediated phosphorylation of PKB/akt, inhibition of apoptosis, and replication of 32D^IR cells during insulin stimulation. These results suggest that a phosphotyrosine-independent mechanism mediated by the PH and PTB domains promoted antiapoptotic and growth actions of insulin. Although PI 3-kinase was not activated, its phospholipid products were required, since LY294002 inhibited these responses. Without IRS-1, a chimeric insulin receptor containing a tail of tyrosine phosphorylation sites derived from IRS-1 activated the PI 3-kinase cascade but failed to inhibit apoptosis. Thus, phosphotyrosine-independent IRS-1-linked pathways may be critical for survival and growth of IL-3-deprived 32D cells during insulin stimulation.     

Different subcellular localization and phosphoinositides binding of insulin receptor substrate protein pleckstrin homology domains

Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3-5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.     

Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor

Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.     

Immunoreceptor tyrosine-based inhibitory motif of the IL-4 receptor associates with SH2-containing phosphatases and regulates IL-4-induced proliferation.

Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R alpha-chain (IL-4Ralpha) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Ralpha by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Ralpha in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Ralpha were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Ralpha. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Ralpha, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5'-phosphatase. The in vivo association of SH2-containing inositol 5'-phosphatase with IL-4Ralpha was verified by coimmunoprecipitation with anti-IL-4Ralpha Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.     

Expression and function of the insulin receptor substrate proteins in cancer

The Insulin Receptor Substrate (IRS) proteins are cytoplasmic adaptor proteins that function as essential signaling intermediates downstream of activated cell surface receptors, many of which have been implicated in cancer. The IRS proteins do not contain any intrinsic kinase activity, but rather serve as scaffolds to organize signaling complexes and initiate intracellular signaling pathways. As common intermediates of multiple receptors that can influence tumor progression, the IRS proteins are positioned to play a pivotal role in regulating the response of tumor cells to many different microenvironmental stimuli. Limited studies on IRS expression in human tumors and studies on IRS function in human tumor cell lines and in mouse models have provided clues to the potential function of these adaptor proteins in human cancer. A general theme arises from these studies; IRS-1 and IRS-4 are most often associated with tumor growth and proliferation and IRS-2 is most often associated with tumor motility and invasion. In this review, we discuss the mechanisms by which IRS expression and function are regulated and how the IRS proteins contribute to tumor initiation and progression.     

Differential modulation of the tyrosine phosphorylation state of the insulin receptor by IRS (insulin receptor subunit) proteins.

In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR+IRS-1F18). Therefore, IRS-1 and IRS-2 appeared to have different effects on insulin receptor phosphorylation and downstream signaling. Preincubation of cells with pervanadate greatly decreased protein-tyrosine phosphatase activity in all four cell lines. After pervanadate treatment, tyrosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1F18 cells was markedly increased, but pervanadate had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to function differently in their effects on signaling downstream of the insulin receptor. IRS-1 may play a major role in regulating insulin receptor phosphorylation and enhancing downstream signaling after insulin stimulation.     

Transgenic expression of insulin receptor substrate 2 in murine B cells alters the cell density-dependence of IgE production in vitro and enhances IgE production in vivo

Previous studies have shown that insulin receptor substrate (IRS)1 and IRS2 mediate proliferative and antiapoptotic signaling through the IL-4R in 32D cells; however their role in regulating normal B cell responses is not clear. To investigate the role of IRS2 in normal B cell function, we developed IRS2 transgenic (Tg) mice on the C57BL/6 background. Western blot analysis revealed a 2-fold elevation in IRS2 protein levels in Tg(+) mice compared with littermate controls and a 3-fold increase in basal tyrosine phosphorylated IRS2 in the absence of IL-4 stimulation. IL-4-induced tyrosine phosphorylation of IRS2 was elevated in Tg(+) B cells, whereas IL-4-induced phosphorylation of STAT6 was similar between Tg(+) and Tg(-) B cells. Tg expression of IRS2 had little effect on IL-4-mediated proliferation and no effect on protection from apoptosis. However, production of IgE and IgG1 by Tg(+) B cells using standard in vitro conditions was diminished 50-60%. Because Ig production in vitro is known to be highly cell concentration-dependent, we performed experiments at different cell concentrations. Interestingly, at very low B cell concentrations (1000-5000 B cells/well), IgE and IgG1 production by Tg(+) B cells was greater than that of controls, whereas at higher cell concentrations (10,000-20,000 cells/well) Ig production by Tg(+) B cells was less than controls. Furthermore, in vivo immunization with OVA-alum or goat anti-IgD resulted in elevated serum IgE levels in the Tg(+) mice. These results indicate that overexpression of IRS2 alters the B cell intrinsic density-dependence of IgE and IgG1 production in vitro and enhances IgE responses in vivo.     

IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis

Insulin receptor substrate (IRS) proteins are tyrosine phosphorylated and mediate multiple signals during activation of the receptors for insulin, insulin-like growth factor 1 (IGF-1), and various cytokines. In order to distinguish common and unique functions of IRS-1, IRS-2, and IRS-4, we expressed them individually in 32D myeloid progenitor cells containing the human insulin receptor (32D(IR)). Insulin promoted the association of Grb-2 with IRS-1 and IRS-4, whereas IRS-2 weakly bound Grb-2; consequently, IRS-1 and IRS-4 enhanced insulin-stimulated mitogen-activated protein kinase activity. During insulin stimulation, IRS-1 and IRS-2 strongly bound p85alpha/beta, which activated phosphatidylinositol (PI) 3-kinase, protein kinase B (PKB)/Akt, and p70(s6k), and promoted the phosphorylation of BAD. IRS-4 also promoted the activation of PKB/Akt and BAD phosphorylation during insulin stimulation; however, it weakly bound or activated p85-associated PI 3-kinase and failed to mediate the activation of p70(s6k). Insulin strongly inhibited apoptosis of interleukin-3 (IL-3)-deprived 32D(IR) cells expressing IRS-1 or IRS-2 but failed to inhibit apoptosis of cells expressing IRS-4. Consequently, 32D(IR) cells expressing IRS-4 proliferated slowly during insulin stimulation. Thus, the activation of PKB/Akt and BAD phosphorylation might not be sufficient to inhibit the apoptosis of IL-3-deprived 32D(IR) cells unless p85-associated PI 3-kinase or p70(s6k) are strongly activated.     

IRS-2 branch of IGF-1 receptor signaling is essential for appropriate timing of myelination

Insulin-like growth factor (IGF)-1 increases proliferation, inhibits apoptosis and promotes differentiation of oligodendrocytes and their precursor cells, indicating an important function for IGF-1 receptor (IGF-1R) signaling in myelin development. The insulin receptor substrates (IRS), IRS-1 and -2 serve as intracellular IGF-1R adaptor proteins and are expressed in neurons, oligodendrocytes and their precursors. To address the role of IRS-2 in myelination, we analyzed myelination in IRS-2 deficient (IRS-2(-/-)) mice and age-matched controls during postnatal development. Interestingly, expression of the most abundant myelin proteins, myelin basic protein and proteolipid protein was reduced in IRS-2(-/-) brains at postnatal day 10 (P10) as compared to controls. myelin basic protein immunostaining in P10-IRS-2(-/-) mice revealed a reduced immunostaining, but an unchanged regional distribution pattern. In cerebral myelin isolates at P10 unaltered relative expression of different myelin proteins was found, indicating quantitatively reduced but not qualitatively altered myelination. Interestingly, up-regulation of IRS-1 expression and increased IGF-1R signaling were observed in IRS-2(-/-) mice at P10-14, indicating a compensatory mechanism to overcome IRS-2 deficiency. Adult IRS-2(-/-) mice showed unaltered myelination and motor function. Furthermore, in neuronal/brain-specific insulin receptor knockout mice myelination was unchanged. Thus, our experiments reveal that IGF-1R/IRS-2 mediated signals are critical for appropriate timing of myelination in vivo.     

Insulin Receptor Substrate Adaptor Proteins Mediate Prognostic Gene Expression Profiles in Breast Cancer

Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer.     

Dual regulation of upstream binding factor 1 levels by IRS-1 and ERKs in IGF-1-receptor signaling

The Upstream Binding Factor 1 (UBF1) is a nucleolar protein that participates in the regulation of RNA polymerase I activity and ribosomal RNA (rRNA) synthesis. In 32D myeloid cells expressing the type 1 insulin-like growth factor receptor (IGF-IR), the UBF1 protein (but not its mRNA) is down regulated when the cells are shifted from Interleukin-3 (IL-3) to IGF-1. Ectopic expression of insulin receptor substrate-1 (IRS-1) in these cells inhibits the down-regulation of UBF1. We now show that the stability of UBF1 in 32D-derived cells requires also a signal from the extracellular regulated kinases (ERKs). When ERKs signaling is defective, as in cells over-expressing the insulin receptor (InR) or selected mutants of the IGF-1R, UBF1 is down-regulated, even in the presence of IRS-1. The down-regulation is corrected by the expression of an activated Ha-ras, which stimulates ERKs activity. Mutations at threonines 117 and 201 of UBF1, known to be phosphorylated by ERKs, cause its down-regulation. However, when IRS-2, instead of IRS-1, is ectopically expressed in 32D InR cells, ERKs phosphorylation is increased and UBF is stabilized. Taken together, these results indicate that in 32D-derived myeloid cells expressing either the IGF-IR or the InR, UBF1 levels are regulated by signaling from both IRS proteins and ERKs.     

Nuclear translocation of insulin receptor substrate-1 by the simian virus 40 T antigen and the activated type 1 insulin-like growth factor receptor

32D cells are a murine hemopoietic cell line that undergoes apoptosis upon withdrawal of interleukin-3 (IL-3) from the medium. 32D cells have low levels of the type 1 insulin-like growth factor (IGF-I) receptor and do not express insulin receptor substrate-1 (IRS-1) or IRS-2. Ectopic expression of IRS-1 delays apoptosis but cannot rescue 32D cells from IL-3 dependence. In 32D/IRS-1 cells, IRS-1 is detectable, as expected, in the cytosol/membrane compartment. The SV40 large T antigen is a nuclear protein that, by itself, also fails to protect 32D cells from apoptosis. Co-expression of IRS-1 with the SV40 T antigen in 32D cells results in nuclear translocation of IRS-1 and survival after IL-3 withdrawal. Expression of a human IGF-I receptor in 32D/IRS-1 cells also results in nuclear translocation of IRS-1 and IL-3 independence. The phosphotyrosine-binding domain, but not the pleckstrin domain, is necessary for IRS-1 nuclear translocation. Nuclear translocation of IRS-1 was confirmed in mouse embryo fibroblasts. These results suggest possible new roles for nuclear IRS-1 in IGF-I-mediated growth and anti-apoptotic signaling.     

Role of IRS and PHIP on insulin-induced tyrosine phosphorylation and distribution of IRS proteins

To analyze the functional differences of the insulin receptor substrate (IRS) family, the N-terminal fragments containing the pleckstrin homology (PH) domains and the phosphotyrosine-binding (PTB) domains of IRS (IRS-N) proteins, as well as intact IRS molecules, were expressed in Cos-1 cells, and insulin-induced tyrosine phosphorylation and subcellular distribution of IRS proteins were analyzed. In contrast to the distinct affinities toward phosphoinositides, these IRS-N fragments non-selectively inhibited insulin-induced tyrosine phosphorylation of IRS-1, IRS-2 and IRS-3, among which IRS3-N was most effective. The mutations of IRS-1 disrupting all the phosphoinositide-binding sites in both the PH and PTB domains significantly but not completely suppressed tyrosine phosphorylation of IRS-1, which was further inhibited by coexpression of all the IRS-N proteins examined. In contrast, the N-terminal PH domain-interacting region (PHIP-N) of PH-interacting protein (PHIP) did not impair tyrosine phosphorylation of either IRS molecule. The analysis using confocal microscopy also demonstrated that all the IRS-N proteins, but not PHIP-N, suppressed targeting of IRS-1 to the plasma membrane in response to insulin. Moreover, the phosphoinositide affinity-disrupting mutations of IRS-1 significantly impaired but did not completely abrogate the insulin-induced translocation of IRS-1 to the plasma membrane, which was further suppressed by IRS1-N overexpression. These findings suggest that both insulin-induced tyrosine phosphorylation and the cell surface targeting of IRS proteins may be regulated in a similar manner through a target molecule common to the members of the IRS family, and distinct from phosphoinositides or PHIP.     

Progesterone receptor-B regulation of insulin-like growth factor-stimulated cell migration in breast cancer cells via insulin receptor substrate-2

Progesterone action contributes to the signaling of many growth factor pathways relevant to breast cancer tumor biology, including the insulin-like growth factor (IGF) system. Previous work has shown that insulin receptor substrate-2 (IRS-2) but not IRS-1 levels were regulated by progestin in progesterone receptor-B (PR-B) isoform expressing MCF-7 cells (C4-12 PR-B). Furthermore, type 1 IGF receptor (IGF1R) signaling via IRS-2 correlated with the increased cell migration observed in a number of breast cancer cell lines. Consequently, in this study, we examined whether the elevation of IRS-2 protein induced by progestin was sufficient to promote IGF-I-stimulated cell motility. Treatment of C4-12 PR-B cells with progestin shifted the balance of phosphorylation from IRS-1 to IRS-2 in response to IGF-I. This shift in IRS-2 activation was associated with enhanced migration in C4-12 PR-B cells pretreated with progestin, but had no effect on cell proliferation or survival. Treatment of C4-12 PR-B cells with RU486, an antiprogestin, inhibited IGF-induced cell migration. Attenuation of IRS-2 expression using small interfering RNA resulted in decreased IGF-stimulated motility. In addition, IRS-2 knockdown resulted in an abrogation of PKB/Akt phosphorylation but not mitogen-activated protein kinase. Consequently, LY294002, a phosphoinositide-3-kinase inhibitor, abolished IGF-induced cell motility in progestin-treated C4-12 PR-B cells. These data show a role for the PR in functionally promoting growth factor signaling, showing that levels of IRS proteins can determine IGF-mediated biology, PR-B signaling regulates IRS-2 expression, and that IRS-2 can mediate IGF-induced cell migration via phosphoinositide-3-kinase in breast cancer cells.     

Insulin receptor substrate 3 (IRS-3) and IRS-4 impair IRS-1- and IRS-2-mediated signaling

To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protein kinase phosphorylation and egr-1 expression ( approximately 1.8- and approximately 2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.     

Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase.

Insulin rapidly stimulates protein synthesis in a wide variety of tissues. This stimulation is associated with phosphorylation of several translational initiation and elongation factors, but little is known about the signaling pathways to these events. To study these pathways, we have used a myeloid progenitor cell line (32D) which is dependent on interleukin 3 but insensitive to insulin because of the very low levels of insulin receptor (IR) and the complete lack of insulin receptor substrate (IRS)-signaling proteins (IRS-1 and IRS-2). Expression of more IR permits partial stimulation of mitogen-activated protein kinase by insulin, and expression of IRS-1 alone mediates insulin stimulation of the 70-kDa S6 kinase (pp70S6K) by the endogenous IR. However, expression of both IR and IRS-1 is required for stimulation of protein synthesis. Moreover, this effect requires activation of phosphatidylinositol 3-kinase (PI3K), as determined by wortmannin inhibition and the use of an IRS-1 variant lacking all Tyr residues except those which activate PI3K. Stimulation of general protein synthesis does not involve activation by IRS-1 of GRB-2-SOS-p21ras or SH-PTP2, since IRS-1 variants lacking the SH2-binding Tyr residues for these proteins are fully active. Nor does it involve pp70S6K, since rapamycin, while strongly inhibiting the synthesis of a small subset of growth-regulated proteins, only slightly inhibits total protein synthesis. Recruitment of mRNAs to the ribosome is enhanced by phosphorylation of eIF4E, the cap-binding protein, and PHAS-I, a protein that specifically binds eIF4E. The behavior of cell lines containing IRS-1 variants and inhibition by wortmannin and rapamycin indicate that the phosphorylation of both proteins requires IRS-1-mediated stimulation of PI3K and pp70S6K but not mitogen-activated protein kinase or SH-PTP2.