Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

Serine phosphorylation of insulin receptor substrate 1 by inhibitor kappa B kinase complex

Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor kappaB kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser(307) (equivalent to Ser(312) in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-alpha or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser(312) in response to tumor necrosis factor-alpha was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J(2) as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser(312). Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser(312) (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways.     

Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.     

S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-(alpha) signaling through IKK2

S6K1 (p70S6K) is a serine kinase downstream from Akt in the insulin signaling pathway that is involved in negative feedback regulation of insulin action. S6K1 is also activated by TNF-alpha, a pro-inflammatory cytokine. However, its role remains to be characterized. In the current study, we elucidated a mechanism for S6K1 to mediate TNF-alpha-induced insulin resistance in adipocytes and hepatocytes. S6K1 was phosphorylated at Thr-389 in response to TNF-alpha. This led to phosphorylation of IRS-1 by S6K1 at multiple serine residues including Ser-270, Ser-307, Ser-636, and Ser-1101 in human IRS-1 (Ser-265, Ser-302, Ser-632, and Ser-1097, in rodent IRS-1). Direct phosphorylation of these sites by S6K1 was observed in an in vitro kinase assay using purified IRS-1 and S6K1. Phosphorylation of all these serines was increased in the adipose tissue of obese mice. RNAi knockdown demonstrated an important role for S6K1 in mediating TNF-alpha-induced IRS-1 inhibition that led to impaired insulin-stimulated glucose uptake in adipocytes. A point mutant of IRS-1 (S270A) impaired association of IRS-1 with S6K1 resulting in diminished phosphorylation of IRS-1 at three other S6K1 phosphorylation sites (Ser-307, Ser-636, and Ser-1101). Expression of a dominant negative S6K1 mutant prevented TNF-induced Ser-270 phosphorylation and IRS-1 protein degradation. Moreover, in IKK2 (but not IKK1)-null cells, TNF-alpha treatment did not result in Thr-389 phosphorylation of S6K1. We present a new mechanism for TNF-alpha to induce insulin resistance that involves activation of S6K by an IKK2-dependent pathway. S6K directly phosphorylates IRS-1 on multiple serine residues to inhibit insulin signaling.     

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.     

Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase C betaII in the diabetic fat tissue

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser(332). However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser(336) on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at Ser(336) and Ser(332) in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser(336). Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCalpha or PKCbetaII isoforms in cells enhanced IRS-1 phosphorylation at Ser(336) and Ser(332), and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser(336). The expression level and activation state of PKCbetaII, but not PKCalpha, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCbetaII were also associated with enhanced phosphorylation of IRS-1 at Ser(336/332) and elevated activity of GSK-3beta. Finally, adenoviral mediated expression of PKCbetaII in adipocytes enhancedphosphorylation of IRS-1 at Ser(336). Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCbetaII and GSK-3 at Ser(336) and Ser(332). Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.     

The insulin receptor substrate (IRS)-1 pleckstrin homology domain functions in downstream signaling

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.     

Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle

c-Jun NH(2)-terminal kinase (JNK) has been shown to negatively regulate insulin signaling through serine phosphorylation of residue 307 within the insulin receptor substrate-1 (IRS-1) in adipose and liver tissue. Using a rat hindlimb suspension model for muscle disuse atrophy, we found that JNK activity was significantly elevated in atrophic soleus muscle and that IRS-1 was phosphorylated on Ser(307) prior to the degradation of the IRS-1 protein. Moreover, we observed a corresponding reduction in Akt activity, providing biochemical evidence for the development of insulin resistance in atrophic skeletal muscle.     

C-reactive protein suppresses insulin signaling in endothelial cells: role of spleen tyrosine kinase

Although few epidemiological studies have demonstrated that C-reactive protein (CRP) is related to insulin resistance, no study to date has examined the molecular mechanism. Here, we show that recombinant CRP attenuates insulin signaling through the regulation of spleen tyrosine kinase (Syk) on small G-protein RhoA, jun N-terminal kinase (JNK) MAPK, insulin receptor substrate-1 (IRS-1), and endothelial nitric oxide synthase in vascular endothelial cells. Recombinant CRP suppressed insulin-induced NO production, inhibited the phosphorylation of Akt and endothelial nitric oxide synthase, and stimulated the phosphorylation of IRS-1 at the Ser307 site in a dose-dependent manner. These events were blocked by treatment with an inhibitor of RhoA-dependent kinase Y27632, or an inhibitor of JNK SP600125, or the transfection of dominant negative RhoA cDNA. Next, anti-CD64 Fcgamma phagocytic receptor I (FcgammaRI), but not anti-CD16 (FcgammaRIIIa) or anti-CD32 (FcgammaRII) antibody, partially blocked the recombinant CRP-induced phosphorylation of JNK and IRS-1 and restored, to a certain extent, the insulin-stimulated phosphorylation of Akt. Furthermore, we identified that recombinant CRP modulates the phosphorylation of Syk tyrosine kinase in endothelial cells. Piceatannol, an inhibitor of Syk tyrosine kinase, or infection of Syk small interference RNA blocked the recombinant CRP-induced RhoA activity and the phosphorylation of JNK and IRS-1. In addition, piceatannol also restrained CRP-induced endothelin-1 production. We conclude that recombinant CRP induces endothelial insulin resistance and dysfunction, and propose a new mechanism by which recombinant CRP induces the phosphorylation of JNK and IRS-1 at the Ser307 site through a Syk tyrosine kinase and RhoA-activation signaling pathway.     

Accumulation of endogenous methylglyoxal impaired insulin signaling in adipose tissue of fructose-fed rats

Increased accumulation of methylglyoxal (MG) has been linked to different insulin resistance states including diabetes and hypertension. In this study, the effects of MG on insulin signaling pathway were investigated. Following 9 weeks of fructose treatment, an insulin resistance state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, high blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. More importantly, we observed a close correlation between the development of insulin resistance and elevated MG level in serum and adipose tissue. Both insulin resistance state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC). When 3T3-L1 adipocytes were treated directly with MG, the impaired insulin signaling was also observed, indicated by decreased insulin-induced insulin-receptor substrate-1 (IRS-1) tyrosine phosphorylation and the decreased kinase activity of phosphatidylinositol (PI) 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In conclusion, the increase in endogenous MG accumulation impairs insulin-signaling pathway and decreases insulin-stimulated glucose uptake in adipose tissue, which may contribute to the development of insulin resistance.     

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.     

Long-term effects of tumor necrosis factor-alpha treatment on insulin signaling pathway in HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB

Tumor necrosis factor-alpha (TNF-alpha) mediated attenuation of insulin signaling pathway is an important cause in several disorders like obesity, obesity linked diabetes mellitus. TNF-alpha actions vary depending upon concentration and time of exposure in various cells. In the present study, the effects of long-term TNF-alpha (1 ng/ml) exposure on the components of insulin signaling pathway in HepG2 and HepG2 cells overexpressing constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB) have been investigated. In parental HepG2 cells, TNF-alpha treatment for 24 h reduced the phosphorylation of Akt1/PKB-alpha and GSK-3beta and under these conditions cells also showed reduced insulin responsiveness in terms of Akt1/PKB-alpha and GSK-3beta phosphorylation. TNF-alpha pre-incubated HepG2-CA-Akt/PKB cells showed lower reduction in Akt1/PKB-alpha and GSK-3beta phosphorylation and insulin responsiveness after 24 h as compared to parental HepG2 cells. We report that the long-term TNF-alpha pre-incubation in both parental HepG2 and HepG2-CA-Akt/PKB-alpha cells leads to the reduction in the levels of IRS-1 without altering the levels of IRS-2. In order to understand the reason for the differential insulin resistance in both the cell types, the effect of long-term TNF-alpha treatment on the proteins upstream to Akt/PKB was investigated. TNF-alpha pre-incubation also showed reduced insulin-stimulated Tyr phosphorylation of insulin receptor (IR-beta) in both the cell types, moreover hyperphosphorylation of IRS-1 at Ser 312 residue was observed in TNF-alpha pre-incubated cells. As hyperphosphorylation of IRS-1 at Ser 312 can induce its degradation, it is possible that reduced insulin responsiveness after long-term TNF-alpha pre-incubation observed in this study is due to the decrease in IRS-1 levels.     

Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) inhibits insulin signal transduction in a variety of cell backgrounds, which might contribute to peripheral insulin resistance. However, because of the large number of potential phosphorylation sites, the mechanism of inhibition has been difficult to determine. One serine residue located near the phosphotyrosine-binding (PTB) domain in IRS-1 (Ser(307) in rat IRS-1 or Ser(312) in human IRS-1) is phosphorylated via several mechanisms, including insulin-stimulated kinases or stress-activated kinases like JNK1. During a yeast tri-hybrid assay, phosphorylation of Ser(307) by JNK1 disrupted the interaction between the catalytic domain of the insulin receptor and the PTB domain of IRS-1. In 32D myeloid progenitor cells, phosphorylation of Ser(307) inhibited insulin stimulation of the phosphatidylinositol 3-kinase and MAPK cascades. These results suggest that inhibition of PTB domain function in IRS-1 by phosphorylation of Ser(307) (Ser(312) in human IRS-1) might be a general mechanism to regulate insulin signaling.     

Phosphorylation of Ser357 of rat insulin receptor substrate-1 mediates adverse effects of protein kinase C-delta on insulin action in skeletal muscle cells

The activation of the protein kinase C (PKC) family of serine/threonine kinases contributes to the modulation of insulin signaling, and the PKC-dependent phosphorylation of insulin receptor substrate (IRS)-1 has been implicated in the development of insulin resistance. Here we demonstrate Ser(357) of rat IRS-1 as a novel PKC-delta-dependent phosphorylation site in skeletal muscle cells upon stimulation with insulin and phorbol ester using Ser(P)(357) antibodies and active and kinase dead mutants of PKC-delta. Phosphorylation of this site was simulated using IRS-1 Glu(357) and shown to reduce insulin-induced tyrosine phosphorylation of IRS-1, to decrease activation of Akt, and to subsequently diminish phosphorylation of glycogen synthase kinase-3. When the phosphorylation was prevented by mutation of Ser(357) to alanine, these effects of insulin were enhanced. When the adjacent Ser(358), present in mouse and rat IRS-1, was mutated to alanine, which is homologous to the human sequence, the insulin-induced phosphorylation of glycogen synthase kinase-3 or tyrosine phosphorylation of IRS-1 was not increased. Moreover, both active PKC-delta and phosphorylation of Ser(357) were shown to be necessary for the attenuation of insulin-stimulated Akt phosphorylation. The phosphorylation of Ser(357) could lead to increased association of PKC-delta to IRS-1 upon insulin stimulation, which was demonstrated with IRS-1 Glu(357). Together, these data suggest that phosphorylation of Ser(357) mediates at least in part the adverse effects of PKC-delta activation on insulin action.     

Hepatitis C virus core protein upregulates serine phosphorylation of insulin receptor substrate-1 and impairs the downstream akt/protein kinase B signaling pathway for insulin resistance

Chronic hepatitis C virus (HCV) infection has a significantly increased prevalence of type 2 diabetes mellitus (T2DM). Insulin resistance is a critical component of T2DM pathogenesis. Several mechanisms are likely to be involved in the pathogenesis of HCV-related insulin resistance. Since we and others have previously observed that HCV core protein activates c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase, we examined the contribution of these pathways to insulin resistance in hepatocytes. Our experimental findings suggest that HCV core protein alone or in the presence of other viral proteins increases Ser(312) phosphorylation of the insulin receptor substrate-1 (IRS-1). Hepatocytes infected with cell culture-grown HCV genotype 1a or 2a displayed a significant increase in the Ser(473) phosphorylation status of the Ser/Thr kinase protein kinase B (Akt/PKB), while Thr(308) phosphorylation was not significantly altered. HCV core protein-mediated Ser(312) phosphorylation of IRS-1 was inhibited by JNK (SP600125) and phosphatidylinositol-3 kinase (LY294002) inhibitors. A functional assay also suggested that hepatocytes expressing HCV core protein alone or infected with cell culture-grown HCV exhibited a suppression of 2-deoxy-d-[(3)H]glucose uptake. Inhibition of the JNK signaling pathway significantly restored glucose uptake despite HCV core expression in hepatocytes. Taken together, our results demonstrated that HCV core protein increases IRS-1 phosphorylation at Ser(312) which may contribute in part to the mechanism of insulin resistance.     

Identification of enhanced serine kinase activity in insulin resistance

Insulin receptor substrate (IRS) proteins play a crucial role as signaling molecules in insulin action. Serine phosphorylation of IRS proteins has been hypothesized as a cause of attenuating insulin signaling. The current study investigated serine kinase activity toward IRS-1 in several models of insulin resistance. An in vitro kinase assay was developed that used partially purified cell lysates as a kinase and glutathione S-transferase fusion proteins that contained various of IRS-1 fragments as substrates. Elevated serine kinase activity was detected in Chinese hamster ovary/insulin receptor (IR)/IRS-1 cells and 3T3-L1 adipocytes chronically treated with insulin, and in liver and muscle of obese JCR:LA-cp rats. It phosphorylated the 526-859 amino acid region of IRS-1, whereas phosphorylation of the 2-516 and 900-1235 amino acid regions was not altered. Phosphopeptide mapping of the 526-859 region of IRS-1 showed three major phosphopeptides (P1, P2, and P3) with different patterns of phosphorylation depending on the source of serine kinase activity. P1 and P2 were strongly phosphorylated when the kinase activity was prepared from insulin-resistant Chinese hamster ovary/IR/IRS-1 cells, weakly phosphorylated by the kinase activity from insulin-resistant 3T3-L1 adipocytes, and barely phosphorylated when the extract was derived from insulin-resistant liver. In contrast, P3 was phosphorylated by the serine kinase activity prepared from all insulin-resistant cells and tissues of animals. P1 and P2 phosphorylation can be explained by mitogen-activated protein kinase activity based on the phosphopeptide map generated by recombinant ERK2. In contrast, mitogen-activated protein kinase failed to phosphorylate the P3 peptide, suggesting that another serine kinase regulates this modification of IRS-1 in insulin-resistant state.     

In vivo phosphorylation of insulin receptor substrate 1 at serine 789 by a novel serine kinase in insulin-resistant rodents

Insulin resistance is a key pathophysiologic feature of obesity and type 2 diabetes and is associated with other human diseases, including atherosclerosis, hypertension, hyperlipidemia, and polycystic ovarian disease. Yet, the specific cellular defects that cause insulin resistance are not precisely known. Insulin receptor substrate (IRS) proteins are important signaling molecules that mediate insulin action in insulin-sensitive cells. Recently, serine phosphorylation of IRS proteins has been implicated in attenuating insulin signaling and is thought to be a potential mechanism for insulin resistance. However, in vivo increased serine phosphorylation of IRS proteins in insulin-resistant animal models has not been reported before. In the present study, we have confirmed previous findings in both JCR:LA-cp and Zucker fatty rats, two genetically unrelated insulin-resistant rodent models, that an enhanced serine kinase activity in liver is associated with insulin resistance. The enhanced serine kinase specifically phosphorylates the conserved Ser(789) residue in IRS-1, which is in a sequence motif separate from the ones for MAPK, c-Jun N-terminal kinase, glycogen-synthase kinase 3 (GSK-3), Akt, phosphatidylinositol 3'-kinase, or casein kinase. It is similar to the phosphorylation motif for AMP-activated protein kinase, but the serine kinase in the insulin-resistant animals was shown not to be an AMP-activated protein kinase, suggesting a potential novel serine kinase. Using a specific antibody against Ser(P)(789) peptide of IRS-1, we then demonstrated for the first time a striking increase of Ser(789)-phosphorylated IRS-1 in livers of insulin-resistant rodent models, indicating enhanced serine kinase activity in vivo. Taken together, these data strongly suggest that unknown serine kinase activity and Ser(789) phosphorylation of IRS-1 may play an important role in attenuating insulin signaling in insulin-resistant animal models.     

Alpha-synuclein overexpression negatively regulates insulin receptor substrate 1 by activating mTORC1/S6K1 signaling

Alpha-synuclein (α-Syn) is a major component of Lewy bodies, a pathological feature of Parkinson's and other neurodegenerative diseases collectively known as synucleinopathies. Among the possible mechanisms of α-Syn-mediated neurotoxicity is interference with cytoprotective pathways such as insulin signaling. Insulin receptor substrate (IRS)-1 is a docking protein linking IRs to downstream signaling pathways such as phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K)1; the latter exerts negative feedback control on insulin signaling, which is impaired in Alzheimer's disease. Our previous study found that α-Syn overexpression can inhibit protein phosphatase (PP)2A activity, which is involved in the protective mechanism of insulin signaling. In this study, we found an increase in IRS-1 phosphorylation at Ser636 and decrease in tyrosine phosphorylation, which accelerated IRS-1 turnover and reduced insulin-Akt signaling in α-Syn-overexpressing SK-N-SH cells and transgenic mice. The mTOR complex (C)1/S6K1 blocker rapamycin inhibited the phosphorylation of IRS-1 at Ser636 in cells overexpressing α-Syn, suggesting that mTORC1/S6K1 activation by α-Syn causes feedback inhibition of insulin signaling via suppression of IRS-1 function. α-Syn overexpression also inhibited PP2A activity, while the PP2A agonist C2 ceramide suppressed both S6K1 activation and IRS-1 Ser636 phosphorylation upon α-Syn overexpression. Thus, α-Syn overexpression negatively regulated IRS-1 via mTORC1/S6K1 signaling while activation of PP2A reverses this process. These results provide evidence for a link between α-Syn and IRS-1 that may represent a novel mechanism for α-Syn-associated pathogenesis.     

SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice

SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr(960) of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-alpha (TNF-alpha), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-alpha induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-alpha target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.     

Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by Serine 312 phosphorylation

Ser/Thr phosphorylation of insulin receptor substrate-1 (IRS-1) is a negative regulator of insulin signaling. One potential mechanism for this is that Ser/Thr phosphorylation decreases the ability of IRS-1 to be tyrosine-phosphorylated by the insulin receptor. An additional mechanism for modulating insulin signaling is via the down-regulation of IRS-1 protein levels. Insulin-induced degradation of IRS-1 has been well documented, both in cells as well as in patients with diabetes. Ser/Thr phosphorylation of IRS-1 correlates with IRS-1 degradation, yet the details of how this occurs are still unknown. In the present study we have examined the potential role of different signaling cascades in the insulin-induced degradation of IRS-1. First, we found that inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin block the degradation. Second, knockout cells lacking one of the key effectors of this cascade, the phosphoinositide-dependent kinase-1, were found to be deficient in the insulin-stimulated degradation of IRS-1. Conversely, overexpression of this enzyme potentiated insulin-stimulated IRS-1 degradation. Third, concurrent with the decrease in IRS-1 degradation, the inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin also blocked the insulin-stimulated increase in Ser(312) phosphorylation. Most important, an IRS-1 mutant in which Ser(312) was changed to alanine was found to be resistant to insulin-stimulated IRS-1 degradation. Finally, an inhibitor of c-Jun N-terminal kinase, SP600125, at 10 microm did not block IRS-1 degradation and IRS-1 Ser(312) phosphorylation yet completely blocked insulin-stimulated c-Jun phosphorylation. Further, insulin-stimulated c-Jun phosphorylation was not blocked by inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin, indicating that c-Jun N-terminal kinase is unlikely to be the kinase phosphorylating IRS-1 Ser(312) in response to insulin. In summary, our results indicate that the insulin-stimulated degradation of IRS-1 via the phosphatidylinositol 3-kinase pathway is in part dependent upon the Ser(312) phosphorylation of IRS-1.