Human Insulin Receptor Substrate-1 Research Articles

The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

Activation of Wnt/β-catenin signaling restores insulin sensitivity in insulin resistant neurons through transcriptional regulation of IRS-1.

Aberrant expression and phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to brain insulin resistance. However, the underlying mechanism remains elusive. The insulin signaling and Wnt/β-catenin signaling are two critical pathways for normal cellular function, which interact in both peripheral tissues and the brain and may contribute to insulin resistance. In this study, we aimed to investigate the regulation of IRS-1 and its downstream insulin signaling by Wnt/β-catenin signaling in primary neurons. We found that the Wnt agonist Wnt3a enhances the insulin signaling in neurons at the basal state via up-regulation of IRS-1. Moreover, Wnt3a up-regulates IRS-1 expression and effectively ameliorates insulin resistance in rat primary neurons induced by chronic high insulin exposure. The insulin-mediated glucose uptake is also stimulated by Wnt3a at both basal and insulin resistant states. We observed that Wnt activation up-regulates IRS-1 gene transcription and the subsequent protein expression in SH-SY5Y cells and rat primary neurons via different means of Wnt/β-catenin signaling activation, including S33Y β-catenin over-expression, CHIR99021 and Wnt3a treatment. We further clarified the molecular mechanism of IRS-1 transcriptional activation by Wnt/β-catenin signaling. The Wnt transcription factor TCF4 binds to the -529bp to -516bp of the human IRS-1 promoter fragment and activates IRS-1 transcription. Overall, these data suggested that Wnt/β-catenin signaling positively regulates IRS-1 and insulin signaling and protects against insulin resistance in neurons.

Porcine insulin receptor substrate 2: molecular cloning, tissues distribution, and functions in hepatocyte and aortic endothelial cells.

Insulin receptor substrate 2 (IRS-2) modulates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which controls the suppression of gluconeogenic genes; IRS-2 is also a critical node of insulin signaling. Because of the high homology between pig and human IRS-2, we investigated the expression pattern and function of porcine IRS-2. QPCR and immunoblotting were used to detect the IRS-2 expression level in different tissues. There were high IRS-2 levels in the cerebral cortex, hypothalamus, and cerebellum in the central nervous system. In peripheral tissues, IRS-2 was expressed at relatively high levels in the liver. Immunohistochemistry analysis revealed that IRS-2 was mainly distributed in the hypothalamus and cerebral cortex. Furthermore, IRS-2 knockdown porcine hepatocytes and porcine aortic endothelial cells (PAECs) were generated. The IRS-2 knockdown induced abnormal expression of genes involved in glycolipid metabolism in hepatocytes and reduced the antiatherosclerosis ability in PAECs. In addition, we disrupted IRS-2 in porcine embryonic fibroblasts (PEFs) using the CRISPR/Cas9 genome editing system, before finally generating IRS-2 knockout embryos by somatic cell nuclear transfer (SCNT). Taken together, our results indicate that IRS-2 might be a valuable target to establish diabetes and vascular disease models in the pig.

Exosomal biomarkers of brain insulin resistance associated with regional atrophy in Alzheimer's disease.

Here, we hypothesized that these exosomal biomarkers associate with brain atrophy in AD. We studied 24 subjects with biomarker-supported probable AD (low CSF Aβ42 ). Exosomes were isolated from plasma, enriched for neural origin using immunoprecipitation for L1CAM, and measured for pSer312 - and p-panTyr-IRS-1 phosphotypes. MPRAGE images were segmented by brain tissue type and voxel-based morphometry (VBM) analysis for gray matter against pSer312 - and p-panTyr-IRS-1 was conducted. Given the regionally variable brain expression of IRS-1, we used the Allen Brain Atlas to make spatial comparisons between VBM results and IRS-1 expression. Brain volume was positively associated with P-panTyr-IRS-1 and negatively associated with pSer312 -IRS-1 in a strikingly similar regional pattern (bilateral parietal-occipital junction, R middle temporal gyrus). This volumetric association pattern was spatially correlated with Allen Human Brain atlas normal brain IRS-1 expression. Exosomal biomarkers of brain IR are thus associated with atrophy in AD as could be expected by their pathophysiological roles and do so in a pattern that reflects regional IRS-1 expression. Furthermore, neural-origin plasma exosomes may recover molecular signals from specific brain regions. Hum Brain Mapp 38:1933-1940, 2017. © 2017 Wiley Periodicals, Inc.

Data in support of FSH induction of IRS-2 in human granulosa cells: Mapping the transcription factor binding sites in human IRS-2 promoter.

Insulin receptor substrate-2 (IRS-2) plays critical role in the regulation of various metabolic processes by insulin and IGF-1. The defects in its expression and/or function are linked to diseases like polycystic ovary syndrome (PCOS), insulin resistance and cancer. To predict the transcription factors (TFs) responsible for the regulation of human IRS-2 gene expression, the transcription factor binding sites (TFBS) and the corresponding TFs were investigated by analysis of IRS-2 promoter sequence using MatInspector Genomatix software (Cartharius et al., 2005 [1]). The ibid data is part of author׳s publication (Anjali et al., 2015 [2]) that explains Follicle stimulating hormone (FSH) mediated IRS-2 promoter activation in human granulosa cells and its importance in the pathophysiology of PCOS. Further analysis was carried out for binary interactions of TF regulatory genes in IRS-2 network using Cytoscape software tool and R-code. In this manuscript, we describe the methodology used for the identification of TFBSs in human IRS-2 promoter region and provide details on experimental procedures, analysis method, validation of data and also the raw files. The purpose of this article is to provide the data on all TFBSs in the promoter region of human IRS-2 gene as it has the potential for prediction of the regulation of IRS-2 gene in normal or diseased cells from patients with metabolic disorders and cancer.

The 14q32 MicroRNA-487b Targets the Antiapoptotic Insulin Receptor Substrate 1 in Hypertension-Induced Remodeling of the Aorta

To study the role of microRNAs in hypertension-induced vascular pathology before the onset of symptoms of severe cardiovascular disease. MicroRNAs play a crucial role in cardiovascular disease. However, microRNAs are often studied in full-blown cardiovascular disease models, not during development of cardiovascular pathology. Angiotensin II was infused into healthy adult rats, inducing chronic hypertension, and microRNA expression profiles were obtained. The most prominently regulated microRNA, miR-487b, was further investigated, using primary cultures of rat aortic and human umbilical cord arterial cells. MiR-487b is predicted to target insulin receptor substrate 1 (IRS1). IRS1 plays an important role in both insulin signaling and cell proliferation and survival. IRS1 mRNA and protein levels were downregulated in aortae of hypertensive rats. MiR-487b binds directly to both rat and human IRS1 3'UTR and inhibits reporter gene expression in vitro. In primary rat and human arterial adventitial fibroblasts, inhibition of miR-487b leads to upregulation of IRS1 expression. Upregulation of miR-487b had the opposite effect, confirming direct targeting of IRS1 by miR-487b.Immunohistochemistry of aortic cross sections and rt/qPCR analyses of the separate aortic wall layers showed that both IRS1 and miR-487b were present mainly in the adventitia and less or not at all in the intima and tunica media. IRS1 expression in adventitial fibroblasts was predominantly nuclear and nuclear IRS1 is known to have antiapoptotic effects. Indeed, inhibition of miR-487b protected adventitial fibroblasts, and also medial smooth muscle cells, against serum starvation-induced apoptosis and increased cell survival. Angiotensin II-induced hypertension leads to upregulation of miR-487b, which targets IRS1. Via downregulation of IRS1, miR-487b can contribute to cell death and loss of adventitial and medial integrity during hypertension-induced vascular pathology.

Resistin Promotes Cardiac Hypertrophy via the AMP-activated Protein Kinase/Mammalian Target of Rapamycin (AMPK/mTOR) and c-Jun N-terminal Kinase/Insulin Receptor Substrate 1 (JNK/IRS1) Pathways

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

Label-free Relative Quantification of Co-eluting Isobaric Phosphopeptides of Insulin Receptor Substrate-1 by HPLC-ESI-MS/MS

Intracellular signal transduction is often regulated by transient protein phosphorylation in response to external stimuli. Insulin signaling is dependent on specific protein phosphorylation events, and analysis of insulin receptor substrate-1 (IRS-1) phosphorylation reveals a complex interplay between tyrosine, serine, and threonine phosphorylation. The phospho-specific antibody-based quantification approach for analyzing changes in site-specific phosphorylation of IRS-1 is difficult due to the dearth of phospho-antibodies compared with the large number of known IRS-1 phosphorylation sites. We previously published a method detailing a peak area-based mass spectrometry approach, using precursor ions for peptides, to quantify the relative abundance of site-specific phosphorylation in the absence or presence of insulin. We now present an improvement wherein site-specific phosphorylation is quantified by determining the peak area of fragment ions respective to the phospho-site of interest. This provides the advantage of being able to quantify co-eluting isobaric phosphopeptides (differentially phosphorylated versions of the same peptide), allowing for a more comprehensive analysis of protein phosphorylation. Quantifying human IRS-1 phosphorylation sites at Ser303, Ser323, Ser330, Ser348, Ser527, and Ser531 shows that this method is linear (n = 3; r(2) = 0.85 +/- 0.05, 0.96 +/- 0.01, 0.96 +/- 0.02, 0.86 +/- 0.07, 0.90 +/- 0.03, 0.91 +/- 0.04, respectively) over an approximate 10-fold range of concentrations and reproducible (n = 4; coefficient of variation = 0.12, 0.14, 0.29, 0.30, 0.12, 0.06, respectively). This application of label-free, fragment ion-based quantification to assess relative phosphorylation changes of specific proteins will prove useful for understanding how various cell stimuli regulate protein function by phosphorylation.

O-Linked N-Acetylglucosamine Modification of Insulin Receptor Substrate-1 Occurs in Close Proximity to Multiple SH2 Domain Binding Motifs

Insulin receptor substrate-1 (IRS-1) is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Ser/Thr kinases impact the metabolic and mitogenic effects elicited by insulin and IGF-1 through feedback and feed forward regulation at the level of IRS-1. Ser/Thr residues of IRS-1 are also O-GlcNAc-modified, which may influence the phosphorylation status of the protein. To facilitate the understanding of the functional effects of O-GlcNAc modification on IRS-1-mediated signaling, we identified the sites of O-GlcNAc modification of rat and human IRS-1. Tandem mass spectrometric analysis of IRS-1, exogenously expressed in HEK293 cells, revealed that the C terminus, which is rich in docking sites for SH2 domain-containing proteins, was O-GlcNAc-modified at multiple residues. Rat IRS-1 was O-GlcNAc-modified at Ser(914), Ser(1009), Ser(1036), and Ser(1041). Human IRS-1 was O-GlcNAc-modified at Ser(984) or Ser(985), at Ser(1011), and possibly at multiple sites within residues 1025-1045. O-GlcNAc modification at a conserved residue in rat (Ser(1009)) and human (Ser(1011)) IRS-1 is adjacent to a putative binding motif for the N-terminal SH2 domains of p85alpha and p85beta regulatory subunits of phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2 (PTPN11). Immunoblot analysis using an antibody generated against human IRS-1 Ser(1011) GlcNAc further confirmed the site of attachment and the identity of the +203.2-Da mass shift as beta-N-acetylglucosamine. The accumulation of IRS-1 Ser(1011) GlcNAc in HEPG2 liver cells and MC3T3-E1 preosteoblasts upon inhibition of O-GlcNAcase indicates that O-GlcNAcylation of endogenously expressed IRS-1 is a dynamic process that occurs at normal glucose concentrations (5 mm). O-GlcNAc modification did not occur at any known or newly identified Ser/Thr phosphorylation sites and in most cases occurred simultaneously with phosphorylation of nearby residues. These findings suggest that O-GlcNAc modification represents an additional layer of posttranslational regulation that may impact the specificity of effects elicited by insulin and IGF-1.

The IRS2 Gly1057Asp Variant Is Associated With Human Longevity

Reduced insulin and insulin-like growth factor-1 (IGF-1) signaling extends the life span of invertebrate and mammals. Recently, reduced insulin receptor substrate-2 (IRS2) signaling was found associated with increased longevity in mice. The aim of our study was to evaluate whether a common polymorphism (Gly1057Asp) in human IRS2 gene is associated with human longevity. Six hundred seventy-seven participants (289 males and 388 females) between 16 and 104 years of age, categorized as long lived (LL; >85 years old) or controls (C; <85 years old), were genotyped for Gly1057Asp-IRS2 locus variability (rs1805097). All participants, contacted at home or in their institution or selected from Italian geriatric and internal medicine or geriatric rehabilitation structures, underwent to a clinical, biochemical, and functional characterization, with particular attention to the insulin and IGF-1 signaling. Insulin resistance (Homeostasis Model Assessment [HOMA]-IR), insulin sensitivity (HOMA IS), and ss-cell function (HOMA-B cell) were calculated by the HOMA2 calculator v2.2 (www.dtu.ox.ac.uk/homa). In the whole population, homozygous IRS2(Asp/Asp) participants were more represented among LL versus C participants (16.7% vs 12.0%; p = .04). The association between IRS2 gene polymorphism with longevity (being LL) was independent of anthropometric and metabolic covariates (odds ratio: 2.07, 95% confidence interval [CI] = 1.38-3.12; p = .001). Categorizing participants into percentiles by age, IRS2(Asp/Asp) participants were more likely to reach extreme old age (>or=90 percentile, 96-104 years; odds ratio: 2.03, 95% CI = 1.39-2.99; p = .0003). These results support the hypothesis that the IRS2 branch of the insulin and IGF signaling is associated with human longevity. Further studies will be necessary for replicating our finding in an independent larger population group with sufficient power before the association between IRS2 gene polymorphism and longevity can be regarded as proven. Furthermore, studies of genetic and/or environmental background interactions may be useful after basic replication is complete.

Expression and localisation of insulin receptor substrate 2 in normal intestine and colorectal tumours. Regulation by intestine-specific transcription factor CDX2

Background and aims:Self-renewal and differentiation of intestinal epithelium is a tightly regulated process, whose perturbations are implicated in human colorectal tumourigenesis. The insulin/insulin-like growth factor (IGF) signalling pathway may play...

Apolipoprotein CIII and Atherosclerosis

The prevailing concept of mechanisms responsible for the development of atherosclerotic lesions largely focuses on the accumulation and retention of low-density lipoproteins in the arterial intima and their subsequent oxidative modification. This oxidation leads to activation of the endothelium, and particularly, expression of adhesion molecules that mediate leukocyte adherence and chemokines which initiate the inflammation reaction that is widely accepted as being responsible for the development and progression of atherosclerotic lesions.1,2 There is also a strong body of evidence to indicate that elevated triacylglycerides (triglycerides) are an independent risk factor for atherosclerosis.3–5 Article p 731 One mechanism that can contribute to elevated triglycerides involves apolipoprotein CIII (apoCIII). apoCIII is a small protein that resides on the surface of very-low-density lipoproteins (VLDLs), low-density lipoproteins, chylomicrons, and high-density lipoproteins (Figure). It exists as multiple species, as either a nonglycosylated isoform (apoCIIIo) or a glycosylated isoform (apoCIII1 or apoCIII2); all three isoforms have similar plasma half-lives and probably have very similar physiological functions. Increased apoCIII production is a characteristic feature of patients with hypertriglyceridemia,6 and plasma apoCIII levels have been positively correlated with plasma triacylglycerol concentrations and also have been associated with severity of hypertriglyceridemia.7 Elevated plasma apoCIII concentration and, specifically, accumulation of apoCIII in triacylglycerol-rich lipoproteins is casually related to hypertriglyceridemia in patients with metabolic syndrome and has also been associated with insulin resistance.8 apoCIII is a major regulator of lipolysis, as it noncompetitively inhibits endothelial-bound lipoprotein lipase, the enzyme that hydrolyzes triacylglycerols in …

IRS-1 transgenic mice show increased epididymal fat mass and insulin resistance

Insulin receptor substrate-1 (IRS-1) is the major substrate of both the insulin receptor and the IGF-1 receptor. In this study, we created IRS-1 transgenic (IRS-1-Tg) mice which express human IRS-1 cDNA under control of the mouse IRS-1 gene promoter. In the IRS-1-Tg mice, IRS-1 mRNA expression was significantly increased in almost all tissues, but its protein expression was increased in very limited tissues (epididymal fat and skeletal muscle). IRS-1-Tg mice showed glucose intolerance and significantly enlarged epididymal fat mass, as well as elevated serum TNF-α concentrations. Importantly insulin signaling was significantly attenuated in the liver of IRS-1-Tg mice, which may contribute to the glucose intolerance. Our results suggest that excess IRS-1 expression may not provide a beneficial impact on glucose homeostasis in vivo.

Phosphorylation of Human Insulin Receptor Substrate-1 at Serine 629 Plays a Positive Role in Insulin Signaling

The function of insulin receptor substrate-1 (IRS-1) is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, whereas phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of human IRS-1 at Ser(629) was increased by insulin in Chinese hamster ovary cells expressing the insulin receptor (1.26 +/- 0.09-fold; P < 0.05) and L6 cells (1.35 +/- 0.29-fold; P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser(629) revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser(629) in cells was decreased upon treatment with either an Akt inhibitor or by coexpression with kinase dead Akt, whereas Ser(629) phosphorylation was increased by coexpression with constitutively active Akt. In addition, Ser(629) of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser(629) by a Ser(629)Ala mutation resulted in increased phosphorylation of Ser(636), a known negative regulator of IRS-1, without affecting phosphorylation of Tyr(632) or Ser(616). Cells expressing the Ser(629)Ala mutation, along with increased Ser(636) phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol 3'-kinase with IRS-1 and decreased phosphorylation of Akt at Ser(473). Finally, in vitro phosphorylation of a Ser(629)-containing IRS-1 fragment with Akt reduces the subsequent ability of ERK to phosphorylate Ser(636/639). These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser(629), resulting in decreased phosphorylation of IRS-1 at Ser(636) and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and, ultimately, the pathogenesis of type 2 diabetes.

Human, but not rat, IRS1 targets to the plasma membrane in both human and rat adipocytes

Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.

Quantification of Phosphorylation of Insulin Receptor Substrate-1 by HPLC-ESI-MS/MS

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.

The Phosphorylation of Ser318 of Insulin Receptor Substrate 1 Is Not per se Inhibitory in Skeletal Muscle Cells but Is Necessary to Trigger the Attenuation of the Insulin-stimulated Signal

The Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS) is one key mechanism to stimulate and/or attenuate insulin signal transduction. Using a phospho-specific polyclonal antibody directed against phosphorylated Ser(318) of IRS-1, we found a rapid and transient insulin-stimulated phosphorylation of Ser(318) in human and rodent skeletal muscle cell models and in muscle tissue of insulin-treated mice. None of the investigated insulin resistance-associated factors (e.g. high glucose, tumor necrosis factor-alpha, adrenaline) stimulated the phosphorylation of Ser(318). Studying the function of this phosphorylation, we found that replacing Ser(318) by alanine completely prevented both the attenuation of insulin-stimulated Akt/protein kinase B Ser(473) phosphorylation and glucose uptake after 60 min of insulin stimulation. Unexpectedly, after acute insulin stimulation, we observed that phosphorylation of Ser(318) is not inhibitory but rather enhances insulin signal transduction because introduction of Ala(318) led to a reduction of the insulin-stimulated Akt/protein kinase B phosphorylation. Furthermore, replacing Ser(318) by glutamate, i.e. mimicking phosphorylation, improved glucose uptake after acute insulin stimulation. These data suggest that phosphorylation of Ser(318) is not per se inhibitory but is necessary to trigger the attenuation of the insulin-stimulated signal in skeletal muscle cells. Investigating the molecular mechanism of insulin-stimulated Ser(318) phosphorylation, we found that phosphatidylinositol 3-kinase-mediated activation of atypical protein kinase C-zeta and recruitment of protein kinase C-zeta to IRS-1 was responsible for this phosphorylation. We conclude that Ser(318) phosphorylation of IRS-1 is an early physiological event in insulin-stimulated signal transduction, which attenuates the continuing action of insulin.

Identification of Insulin Receptor Substrate 1 Serine/Threonine Phosphorylation Sites Using Mass Spectrometry Analysis: Regulatory Role of Serine 1223

Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation.

Shp2 Is Required for Protein Kinase C-dependent Phosphorylation of Serine 307 in Insulin Receptor Substrate-1

The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), we detected only Ser-318, but not Ser-307 phosphorylation, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. As we have observed recently that the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, we studied the role of Shp2 in this context. We found that phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depended on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 were blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1.

Human Insulin Receptor Substrate-1 (IRS-1) Polymorphism G972R Causes IRS-1 to Associate with the Insulin Receptor and Inhibit Receptor Autophosphorylation

The most commonly detected polymorphism in human insulin receptor substrate-1 (IRS-1), a glycine to arginine change at codon 972 (G972R), is associated with an increased risk of Type 2 diabetes and insulin resistance. To determine the molecular mechanism by which this polymorphism may be linked to insulin resistance, we produced recombinant peptides comprising amino acid residues 925-1008 from IRS-1 that contain either a glycine or arginine at codon 972 and the two nearby tyrosine phosphorylation consensus sites (EY(941)MLM and DY(989)MTM), which are known binding sites for the p85alpha regulatory subunit of phosphatidylinositol 3-kinase. The wild type peptide could be phosphorylated at these sites in vitro by purified insulin receptor. Introduction of the G972R polymorphism into the peptide reduced the amount of tyrosine phosphorylation by >60%. Pull-down experiments indicated that there was an association between the IRS-1-(925-1008) peptide and the insulin receptor that was markedly enhanced by the presence of the G972R polymorphism. The use of additional overlapping fragments localized this interaction to domains between residues 950-986 of IRS-1 and residues 966-1271 of the insulin receptor, containing the tyrosine kinase domain of the receptor. In addition, the IRS-1-(925-1008) G972R peptide acted as a competitive inhibitor of insulin receptor and insulin-like growth factor-1 receptor autophosphorylation. Taken together, these data indicate that the G972R naturally occurring polymorphism of IRS-1 not only reduces phosphorylation of the substrate but allows IRS-1 to act as an inhibitor of the insulin receptor kinase, producing global insulin resistance.