Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion μLC/ES-API-CID-MS hybrid scan technique

Beck, Alexander; Moeschel, Klaus; Deeg, Martin; Häring, Hans Ulrich; Voelter, Wolfgang; Schleicher, Erwin; Lehmann, Rainer

doi:10.1016/s1044-0305(03)00122-3

Cited by 28 publications

(26 citation statements)

References 23 publications

(10 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…357 of IRS-1 in vitro by Greene et al (41) and our group using the previously described mass spectrometry-based identification procedure (43). The sequence homology of rodent and human IRS-1 is shown in Fig.…”

Section: Identification Of Ser 357 As a Novel Phosphorylation Site Inmentioning

confidence: 96%

Phosphorylation of Ser357 of Rat Insulin Receptor Substrate-1 Mediates Adverse Effects of Protein Kinase C-δ on Insulin Action in Skeletal Muscle Cells

Waraich

Weigert

Kalbacher

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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-␦-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-␦. 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 To accomplish its fundamental role in maintaining in vivo metabolic homeostasis, insulin binds and activates insulin receptors, which in turn recruit insulin-receptor substrate (IRS) 2 proteins (1). By disruption of their respective genes in mice, IRS-1 and IRS-2 have been assigned a central role in mediating the normal actions of insulin. Defects at the level of the IRS proteins also comprise a major locus for the development of metabolic disorders, including insulin resistance and type 2 diabetes (2, 3). IRS proteins are regulated at several steps, including gene expression, protein degradation, and phosphorylation (4, 5). Although the phosphorylation of IRS-1 on tyrosine residues is mandatory for insulin-stimulated responses, serine/threonine phosphorylation appears to be the mechanism for the precise regulation and could either enhance or attenuate the insulin effects (6, 7). However, in most studies serine phosphorylation of IRS-1 has been implicated as a negative regulator of insulin signaling (8 -12). It can induce the dissociation of IRS-1 from its receptor and hinder the phosphorylation of tyrosine residues (8), release the IRS-1 from intracellular complexes that maintain them in close proximity to the receptor (13), or induce its protein degradation (14). These multiple effects suggest that the serine residues subjected to phosphorylation play a pivotal role in regulating IRS-1 function. Of note, the unbalanced chronic stimulation of IRS-1 serine kinases leads to hyperphosphorylation of IRS-1 and is a major pathophysiological mechanism in the development of insulin resistance. Among these IRS-1 kinases, members of the protein kinase C (PKC) family of serine/threonine kinases have received considerable attention for their regulatory role in insulin signaling. Although particularly the activation of atypical PKCs has been reported as positive modulation of insulin signaling (15, 16), the majority of studies has demonstrated that PKCs are implicated in impaired insulin signaling including classical (17-19), novel (20), and more recently also atypical PKC isoforms (21). Activation of PKC isoforms by insulin (11, 21, 22), hyperglycemia (1...

show abstract

Section: Identification Of Ser 357 As a Novel Phosphorylation Site Inmentioning

confidence: 96%

Phosphorylation of Ser357 of Rat Insulin Receptor Substrate-1 Mediates Adverse Effects of Protein Kinase C-δ on Insulin Action in Skeletal Muscle Cells

Waraich

Weigert

Kalbacher

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Site of PKC-in IRS-1-By developing a novel mass spectrometric approach for phosphopeptide screening out of complex phosphoprotein digest mixtures, we recently identified Ser 318 as a PKC-in vitro phosphorylation site in the IRS-1 N2 fragment (IRS-1 sequence from amino acid 265-522) (14). To search for further in vitro PKC-phosphorylation sites in IRS-1 not covered by the N2-fragment, the PKC--catalyzed 32 P-incorporation in the NH 2 terminal 1 (IRS-1-N1), NH 2 terminal 2 (IRS-1-N2), the middle (IRS-1-M), and the C-terminal part (IRS-1-C) were compared ( Fig.…”

Section: Identification Of the Major In Vitro Phosphorylationmentioning

confidence: 99%

“…1C). According to our previously published procedure (14), mass spectrometric peptide sequencing revealed that more than 85% of total incorporated 32 P-radioactivity is based upon peaks of different variants of the monophosphorylated phospho-Ser 318 . Consequently, these peaks were not 32 P-phosphorylated in the HPLC-separated, mutated GST-Ala 318 -IRS-1 N2 digest.…”

Section: Identification Of the Major In Vitro Phosphorylationmentioning

confidence: 99%

“…Based on our novel previously described mass spectrometric procedure for phosphopeptide screening (14), we identified Ser 318 as the major site phosphorylated by PKC-. Using polyclonal phospho-site-specific antibodies, we demonstrated that this site is also phosphorylated by PKC-in cellular extracts.…”

mentioning

confidence: 99%

“…In-gel Digestion-The GST-IRS-1 N2 -band at 54 kDa was excised and in-gel-digested as described elsewhere (14). The resulting peptides were separated using a Shimadzu HPLC-system with a C2/C18, 100 ϫ 21-mm column (Amersham Biosciences); Detection was as follows: UV at ϭ 214 nm; flow rate ϭ 0.1 ml/min; buffer A, 0.05% trifluoroacetic acid in water (v/v); buffer B, 80% acetonitrile/0.05% trifluoroacetic acid (v/v); gradient, 0% buffer B (10 min), 10% buffer B (20 min), 40% buffer B (60 min), 100% buffer B (90 min), and return to 0% buffer B in 6 min.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Protein Kinase C-ζ-induced Phosphorylation of Ser318 in Insulin Receptor Substrate-1 (IRS-1) Attenuates the Interaction with the Insulin Receptor and the Tyrosine Phosphorylation of IRS-1

Moeschel¹,

Beck²,

Weigert³

et al. 2004

Journal of Biological Chemistry

Self Cite

114

View full text Add to dashboard Cite

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...

show abstract

Solvatochromism and Nonradiative Decay of Intramolecular Charge‐Transfer Excited States: Bands‐of‐Energy Model, Thermodynamics, and Self‐Organization

Pavlovich

2012

ChemPhysChem

View full text Add to dashboard Cite

The fluctuations of orientation and induction interactions in solution and their impact on the broadening of absorption and fluorescence spectra are considered in terms of a bands-of-energy model. Also covered is the application of principles of thermodynamics and self-organization of systems for calculation of solvatochromic shift, among them a component owing to the work on electronic polarization of solvent at the instant of electronic transition in the solute. The findings on solvatochromic shift and spectral broadening open the way to the calculation of solvent effects on the rate constant of nonradiative transitions. As demonstrated herein for 15 fluorophores, the novel theory of nonradiative decay of the intramolecular charge-transfer excited states is carried out for dyes and organic compounds of different nature, both for polar and nonpolar media.

show abstract

Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion μLC/ES-API-CID-MS hybrid scan technique

Cited by 28 publications

References 23 publications

Phosphorylation of Ser357 of Rat Insulin Receptor Substrate-1 Mediates Adverse Effects of Protein Kinase C-δ on Insulin Action in Skeletal Muscle Cells

Phosphorylation of Ser357 of Rat Insulin Receptor Substrate-1 Mediates Adverse Effects of Protein Kinase C-δ on Insulin Action in Skeletal Muscle Cells

Protein Kinase C-ζ-induced Phosphorylation of Ser318 in Insulin Receptor Substrate-1 (IRS-1) Attenuates the Interaction with the Insulin Receptor and the Tyrosine Phosphorylation of IRS-1

Solvatochromism and Nonradiative Decay of Intramolecular Charge‐Transfer Excited States: Bands‐of‐Energy Model, Thermodynamics, and Self‐Organization

Contact Info

Product

Resources

About