Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-, we identified Ser 318 of rat IRS-1 (Ser 323 in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser 318 to alanine). To monitor phosphorylation of Ser 318 in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK IR ). Using the phospho-Ser 318 -specific antibody we observed that insulin stimulates phosphorylation of Ser 318 in IRS-1, which is mediated, at least partially, by PKC-. Moreover, we found that the previously described insulin-stimulated, PKC--mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser 318 to alanine. These results, generated in BHK IR cells, suggest that phosphorylation of Ser 318 by PKC-might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.Insulin resistance is associated with a variety of physiological and patho-physiological states, including type 2 diabetes, hypertension, glucose intolerance, and obesity; however, the molecular mechanisms that modulate insulin signaling under these conditions are difficult to resolve. One important early site of divergence in insulin signaling, which seems to be a relevant target for modulation of the signal, is insulin receptor substrate 1 (IRS-1) 1 (1). IRS-1 is a hydrophilic protein with an apparent molecular mass of 180 kDa containing a conserved pleckstrin homology domain located at the amino terminus, adjacent to a phosphotyrosine binding (PTB) domain (2). After insulin-stimulation, this domain interacts with the activated insulin receptor (IR), IRS-1 is subsequently tyrosine-phosphorylated, and the signal is transmitted further (1, 2).In addition to tyrosine phosphorylation, IRS-1 undergoes Ser/Thr phosphorylation at multiple sites (3, 4). Many in vitro and in vivo studies (5-13) have shown that increased Ser/Thr phosphorylation of IRS-1, e.g. after treatment of cells with either activators of protein kinase C (PKC), Ser/Thr phosphatase inhibitors, high insulin concentrations, or activation of cellular stress pathways by tumor necrosis factor ␣ and other cytokines, inhibits the IR-mediated tyrosine phosphorylation of IRS-1, thereby affecting insulin signal transduction.The atypical PKC-, which is activated by insulin, has been identified to transduce insulin signaling downstream from IRS-1 and phosphatidylinositol 3-kinase in mediating glucose uptake. However, as reported recently, pr...
Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine-318 as a protein kinase C (PKC)-dependent phosphorylation site in Irs1 (Ser-318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity-related insulin resistance, promotes phosphorylation of Ser-318 in Irs1 by a janus kinase 2, Irs2, and PKC-dependent pathway. Mutation of Ser-318 to alanine abrogates the inhibitory effect of leptin on insulin-induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser-318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet-induced obesity Ser-318 phosphorylation levels were already up-regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser-318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser-318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser-318 in Irs1 mediates the inhibitory signal of leptin on the insulin-signaling cascade in obese subjects.
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 resistanceassociated factors (e.g. high glucose, tumor necrosis factor-␣, 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 insulinstimulated 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-and recruitment of protein kinase C-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.
A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.
Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrosprayatmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylationspecific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS 3 . Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-phosphorylation site in the insulin receptor substrate Ϫ1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins. (J Am Soc Mass Spectrom 2003, 14, 401-405)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.