Effect of phosphotyrosyl-IRS-1 level and insulin receptor tyrosine kinase activity on insulin-stimulated phosphatidylinositol 3, MAP,and S6 kinase activities

The role of tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) was studied utilizing parental CHO cells or CHO cells that overexpress IRS-1, the insulin receptor, or both IRS-1 and the insulin receptor. Insulin stimulation of these four cell lines led to progressive levels of IRS-1 tyrosine phosphorylation of one, two, four, and tenfold. Maximal insulin-stimulated IRS-1 associated Ptdlns 3′-kinase activit in these cells was 1-, 1.5-, 3-, and 3-fold, while insulin sensitivity, as determined by ED₅₀, was 1-, 2.5-, 10-, and 10-fold. Both sensitivity and maximal response paralleled the increased level of phosphotyrosyl-IRS-1; however, the increased level of phosphotyrosyl-IRS-1 seen in CHO/IR/IRS-1 cells did not further increase these responses. Likewise, maximal insulin-stimulated MAP kinase activity in these cell lines increased in parallel with IRS-1 tyrosine phosphorylation except in the CHO/IR/IRS-1 cell lines with activity levels of one-, five-, nine-, and ninefold. However, insulin sensitivity of the MAP and S6 kinases and maximal insulin-stimulated S6 kinase activity was not changed by a twofold increase in phosphotyrosyl-IRS-1, but an increase was observed with insulin-stimulated receptor autophosphorylation and kinase activity in CHO/IR cells which led to a tenfold increase in insulin receptor autophosphorylation and a fourfold increase in IRS-1 tyrosine phosphorylation. Thus, these three kinase activities may be differentially coupled to the activation of the insulin receptor kinase activity via IRS-1 and other possible cellular substrates. © 1995 Wiley-Liss, Inc.     

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.     

Insulin receptor substrate 1 rescues insulin action in CHO cells expressing mutant insulin receptors that lack a juxtamembrane NPXY motif.

Insulin signals are mediated through tyrosine phosphorylation of specific proteins such as insulin receptor substrate 1 (IRS-1) and Shc by the activated insulin receptor (IR). Phosphorylation of both proteins is nearly abolished by an alanine substitution at Tyr-960 (A960) in the beta-subunit of the receptor. However, overexpression of IRS-1 in CHO cells expressing the mutant receptor (A960 cells) restored sufficient tyrosine phosphorylation of IRS-1 to rescue IRS-1/Grb-2 binding and phosphatidylinositol 3' kinase activation during insulin stimulation. Shc tyrosine phosphorylation and its binding to Grb-2 were impaired in the A960 cells and were unaffected by overexpression of IRS-1. Although overexpression of IRS-1 increased IRS-1 binding to Grb-2, ERK-1/ERK-2 activation was not rescued. These data suggest that signaling molecules other than IRS-1, perhaps including Shc, are critical for insulin stimulation of p21ras. Interestingly, overexpression of IRS-1 in the A960 cells restored insulin-stimulated mitogenesis and partially restored insulin stimulation of glycogen synthesis. Thus, IRS-1 tyrosine phosphorylation is sufficient to increase the mitogenic response to insulin, whereas insulin stimulation of glycogen synthesis appears to involve other factors. Moreover, IRS-1 phosphorylation is either not sufficient or not involved in insulin stimulation of ERK.     

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.     

The insulin receptor functions normally in Chinese hamster ovary cells after truncation of the C terminus.

We studied the structure and function of the human insulin receptor (IR) and a mutant which lacked the last 43 amino acids of the beta-subunit (IR delta ct). This deletion removed tyrosine (Tyr1322, Tyr1316) and threonine (Thr1336) phosphorylation sites. In Chinese hamster ovary (CHO) cells, insulin binding to the mutant receptor was normal, and [35S]methionine labeling indicated that both the IR and IR delta ct were processed normally; however, the beta-subunit of IR delta ct was 5 kDa smaller than that of the IR. The time course of insulin-stimulated autophosphorylation of the partially purified IR delta ct was normal, but the maximum autophosphorylation was reduced 20-30%. Tryptic phosphopeptide mapping confirmed the absence of the C-terminal phosphorylation sites and indicated that phosphorylation of the regulatory region (Tyr1146, Tyr1150, Tyr1151) occurred normally; kinase activity of the IR and IR delta ct was activated normally by insulin-stimulated autophosphorylation. In the intact CHO cells, insulin-stimulated serine and threonine phosphorylation of the IR delta ct was reduced 20%, suggesting that most Ser/Thr phosphorylation sites are located outside of the C terminus. During insulin stimulation, the wild-type and mutant insulin receptor activated the phosphatidylinositol 3-kinase. Moreover, insulin itself or human-specific anti-insulin receptor antibodies stimulated glycogen and DNA synthesis equally in both CHO/IR and CHO/IR delta ct cells. These data suggest that the C terminus plays a minimal role in IR function and signal transmission in CHO cells.     

JAK1-dependent phosphorylation of insulin receptor substrate-1 (IRS-1) is inhibited by IRS-1 serine phosphorylation.

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) reduces its ability to act as an insulin receptor substrate and inhibits insulin receptor signal transduction. Here, we report that serine phosphorylation of IRS-1 induced by either okadaic acid (OA) or chronic insulin stimulation prevents interferon-alpha (IFN-alpha)-dependent IRS-1 tyrosine phosphorylation and IFN-alpha-dependent IRS-1/phosphatidylinositol 3'-kinase (PI3K) association. In addition, we demonstrate that serine phosphorylation of IRS-1 renders it a poorer substrate for JAK1 (Janus kinase-1). We found that treatment of U266 cells with OA induced serine phosphorylation of IRS-1 and completely blocked IFN-alpha-dependent tyrosine phosphorylation of IRS-1 and IFN-alpha-dependent IRS-1/PI3K association. Additionally, IRS-1 from OA-treated cells could not be phosphorylated in vitro by IFN-alpha-activated JAK1. Chronic treatment of U266 cells with insulin led to a 50% reduction in IFN-alpha-dependent tyrosine phosphorylation of IRS-1 and IRS-1/PI3K association. More importantly, serine-phosphorylated IRS-1-(511-722) could not be phosphorylated in vitro by IFN-alpha-activated JAK1. Taken together, these data indicate that serine phosphorylation of IRS-1 prevents its subsequent tyrosine phosphorylation by JAK1 and suggest that IRS-1 serine phosphorylation may play a counter-regulatory role in pathways outside the insulin signaling system.     

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.     

Positive and negative regulatory role of insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) serine/threonine phosphorylation

Insulin receptor substrates (IRS) 1 and 2 are phosphorylated on serine/threonine (Ser/Thr) residues in quiescent cells (basal phosphorylation), and phosphorylation on both Ser/Thr and tyrosine residues is increased upon insulin stimulation. To determine whether basal Ser/Thr phosphorylation of IRS proteins influences insulin receptor catalyzed tyrosine phosphorylation, recombinant FLAG epitope-tagged IRS-1 (F-IRS-1) and IRS-2 (F-IRS-2) were expressed, purified, and subjected to both dephosphorylation and hyperphosphorylation prior to phosphorylation by the insulin receptor kinase. As expected, hyperphosphorylation of F-IRS-1 and F-IRS-2 by GSK3beta decreased their subsequent phosphorylation on tyrosine residues by the insulin receptor. Surprisingly, however, dephosphorylation of the basal Ser/Thr phosphorylation sites impaired subsequent phosphorylation on tyrosine, suggesting that basal Ser/Thr phosphorylation of F-IRS-1 and F-IRS-2 plays a positive role in phosphorylation by the insulin receptor tyrosine kinase. Dephosphorylation of basal Ser/Thr sites on F-IRS-1 also significantly reduced tyrosine phosphorylation by the IGF-1 receptor. However, dephosphorylation of F-IRS-2 significantly increased phosphorylation by the IGF-1 receptor, suggesting that basal phosphorylation of IRS-2 has divergent effects on its interaction with the insulin and IGF-1 receptors. Phosphorylation of endogenous IRS-1 and IRS-2 from 3T3-L1 adipocytes was modulated in a similar manner. IRS-1 and IRS-2 from serum-fed cells were hyperphosphorylated, and dephosphorylation induced either by serum deprivation or by alkaline phosphatase treatment after immunoprecipitation led to an increase in tyrosine phosphorylation by the insulin receptor. Dephosphorylation of IRS-1 and IRS-2 immunoprecipitated from serum-deprived cells, however, resulted in inhibition of tyrosine phosphorylation by the insulin receptor. These data suggest that Ser/Thr phosphorylation can have both a positive and a negative regulatory role on tyrosine phosphorylation of IRS-1 and IRS-2 by insulin and IGF-1 receptors.     

Phosphatidylinositol 3''-kinase is activated by association with IRS-1 during insulin stimulation.

IRS-1 undergoes rapid tyrosine phosphorylation during insulin stimulation and forms a stable complex containing the 85 kDa subunit (p85) of the phosphatidylinositol (PtdIns) 3'-kinase, but p85 is not tyrosyl phosphorylated. IRS-1 contains nine tyrosine phosphorylation sites in YXXM (Tyr-Xxx-Xxx-Met) motifs. Formation of the IRS-1-PtdIns 3'-kinase complex in vitro is inhibited by synthetic peptides containing phosphorylated YXXM motifs, suggesting that the binding of PtdIns 3'-kinase to IRS-1 is mediated through the SH2 (src homology-2) domains of p85. Furthermore, overexpression of IRS-1 potentiates the activation of PtdIns 3-kinase in insulin-stimulated cells, and tyrosyl phosphorylated IRS-1 or peptides containing phosphorylated YXXM motifs activate PtdIns 3'-kinase in vitro. We conclude that the binding of tyrosyl phosphorylated IRS-1 to the SH2 domains of p85 is the critical step that activates PtdIns 3'-kinase during insulin stimulation.     

Interleukin-1alpha inhibits insulin signaling with phosphorylating insulin receptor substrate-1 on serine residues in 3T3-L1 adipocytes

Proinflammatory cytokines are recently reported to inhibit insulin signaling causing insulin resistance. IL-1alpha is also one of the proinflammatory cytokines; however, it has not been clarified whether IL-1alpha may also cause insulin resistance. Here, we investigated the effects of IL-1alpha treatment on insulin signaling in 3T3-L1 adipocytes. IL-1alpha treatment up to 4 h did not alter insulin-stimulated insulin receptor tyrosine phosphorylation, whereas tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the association with phosphatidylinositol 3-kinase were partially inhibited with the maximal inhibition in around 15 min. IRS-1 was transiently phosphorylated on some serine residues around 15 min after IL-1alpha stimulation, when several serine kinases, IkappaB kinase, c-Jun-N-terminal kinase, ERK, and p70S6K were activated. Chemical inhibitors for these kinases inhibited IL-1alpha-induced serine phosphorylation of IRS-1. Tyrosine phosphorylation of IRS-1 was recovered only by the IKK inhibitor or JNK inhibitor, suggesting specific involvement of these two kinases. Insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake were not inhibited only by IL-1alpha. Interestingly, Akt phosphorylation was synergistically inhibited by IL-1alpha in the presence of IL-6. Taken together, short-term IL-1alpha treatment transiently causes insulin resistance at IRS-1 level with its serine phosphorylation. IL-1alpha may suppress insulin signaling downstream of IRS-1 in the presence of other cytokines, such as IL-6.     

Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling

Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser(302) to study IRS-1-mediated signaling during insulin and insulin-like growth factor IGF-I stimulation. Insulin or IGF-I stimulated phosphorylation of Ser(302) in various cell backgrounds and in murine muscle. Wortmannin or rapamycin inhibited Ser(302) phosphorylation, and amino acids or glucose stimulated Ser(302) phosphorylation, suggesting a role for the mTOR cascade. The Ser(302) kinase associates with IRS-1 during immunoprecipitation, but its identity is unknown. The NH(2)-terminal c-Jun kinase did not phosphorylate Ser(302). Replacing Ser(302) with alanine significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and p85 binding and reduced insulin-stimulated phosphorylation of p70(S6K), ribosomal S6 protein, and 4E-BP1; however, this mutation had no effect on insulin-stimulated Akt or glycogen synthase kinase 3beta phosphorylation. Replacing Ser(302) with alanine reduced insulin/IGF-I-stimulated DNA synthesis. We conclude that Ser(302) phosphorylation integrates nutrient availability with insulin/IGF-I signaling to promote mitogenesis and cell growth.     

Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin

Protein kinase C-zeta (PKC-zeta) is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta. Under in vitro conditions, wild-type PKC-zeta (but not kinase-deficient mutant PKC-zeta) significantly phosphorylated IRS-1. This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A. In addition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [(32)P]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-zeta, and this association was increased 2-fold upon insulin stimulation. Furthermore, the overexpression of PKC-zeta in NIH-3T3(IR) cells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-1-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-zeta. Because PKC-zeta is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-zeta may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.     

Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.     

Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.     

The subcellular fractionation properties and function of insulin receptor substrate-1 (IRS-1) are independent of cytoskeletal integrity

Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, individually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim(-/-) cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton.     

Early insulin signaling cascade in a model of oxidative skeletal muscle: mouse Sol8 cell line

Cell models provide important tools to investigate the mechanisms modulating the insulin-signaling cascade. Insulin interaction and subsequent signaling of cells is complex and regulated at multiple levels: receptor abundance, binding dynamics, phosphorylation/dephosphorylation of tyrosine and serine/threonine residues, and subsequent interactions of key intracellular messengers. We report early insulin signaling events in the mouse Sol8 myogenic cell line. Sol8 cells responded to insulin by increasing total IRS-1, p85 PI3-kinase and tyrosine phosphorylated IRS-1 (pY-IRS-1) at 10 min (P<0.05), but not at 1 min of insulin stimulation. The dose-response relationships at 10-min insulin (10 to 300 nM) stimulation showed that IRS-1 and pY-IRS-1 responded to 100 and 300 nM insulin, and the p85 PI3-kinase response peaked at 30 nM insulin. PI3-kinase appeared to be present in high abundance and, in response to insulin, recruitment to the insulin receptor tyrosine kinase (IR) of IRS-1 and PI3-kinase was observed. The increase in IRS-1 detected in IR immunoprecipitates was twofold, while the corresponding increase in PI3-kinase was threefold, suggesting direct recruitment of PI3-kinase to the IR. PI3-kinase detected in IRS-1 immunoprecipitates in response to insulin increased 1.7-fold. An ultimate target of this pathway, GLUT4 recruitment to the PM, was delayed (30 min), the increase in GLUT4 being of similar magnitude (1.6-fold) to the early signaling events. Saturation binding analysis indicated that IR in the plasma membrane was not down-regulated in response to insulin. The present study suggests that early signaling events in the insulin cascade are invoked in Sol8 myogenic cells and that this cell line provides a useful model to study insulin signaling.     

Frap-dependent serine phosphorylation of IRS-1 inhibits IRS-1 tyrosine phosphorylation

We have previously shown that interferon-alpha (IFN alpha)-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is impaired by serine phosphorylation of IRS-1 due to the reduced ability of serine phosphorylated IRS-1 to serve as a substrate for Janus kinase 1 (JAK1). Here we report that FKBP12-rapamycin-associated protein (FRAP) is a physiologic IRS-1 kinase that blocks IFN alpha signaling by serine phosphorylating IRS-1. We found that both FRAP and insulin-activated p70 S6 kinase (p70(s6k)) serine phosphorylated IRS-1 between residues 511 and 772 (IRS-1(511-772)). Importantly, only FRAP-dependent IRS-1(511-772) serine phosphorylation inhibited by 50% subsequent JAK1-dependent tyrosine phosphorylation of IRS-1. Furthermore, treatment of U266 cells with the FRAP inhibitor rapamycin increased IFN alpha-dependent tyrosine phosphorylation by twofold while reducing constitutive IRS-1 serine phosphorylation within S/T-P motifs by 80%. Taken together, these data indicate that FRAP, but not p70(s6k), is a likely physiologic IRS-1 serine kinase that negatively regulates JAK1-dependent IRS-1 tyrosine phosphorylation and suggests that FRAP may modulate IRS-dependent cytokine signaling.     

Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.

We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.     

Serine phosphorylation proximal to its phosphotyrosine binding domain inhibits insulin receptor substrate 1 function and promotes insulin resistance

Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-1(7A)), unlike wild-type IRS-1 (IRS-1(WT)), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-1(7A) to remain complexed with the insulin receptor (IR), unlike IRS-1(WT), which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-1(7A) and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.     

Role of IRS-1-GRB-2 complexes in insulin signaling.

GRB-2 is a small SH2- and SH3 domain-containing adapter protein that associates with the mammalian SOS homolog to regulate p21ras during growth factor signaling. During insulin stimulation, GRB-2 binds to the phosphorylated Y895VNI motif of IRS-1. Substitution of Tyr-895 with phenylalanine (IRS-1F-895) prevented the IRS-1-GRB-2 association in vivo and in vitro. The myeloid progenitor cell line, 32-D, is insensitive to insulin because it contains few insulin receptors and no IRS-1. Coexpression of IRS-1 or IRS-1F-895 with the insulin receptor was required for insulin-stimulated mitogenesis in 32-D cells, while expression of the insulin receptor alone was sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p21ras and mitogen-activated protein (MAP) kinase. The Shc-GRB-2 complex formed during insulin stimulation is a possible mediator of p21ras and MAP kinase activation in IRS-1-deficient 32-D cells. Interestingly, IRS-1, but not IRS-1F-895, enhanced the stimulation of MAP kinase by insulin in 32-D cells expressing insulin receptors. Thus, IRS-1 contributes to the stimulation of MAP kinase by insulin, probably through formation of the IRS-1-GRB-2 complex at Tyr-895. Our results suggest that the Shc-GRB-2 complex and the activation of p21ras-dependent signaling pathways, including MAP kinase, are insufficient for insulin-stimulated mitogenesis and that the essential function(s) of IRS-1 in proliferative signaling is largely unrelated to IRS-1-GRB-2 complex formation.