title: In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.
abstract: Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.
authors.affiliation: Department of Clinical Biochemistry and Pathobiochemistry, German Diabetes Research Institute, Germany.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Sommerfeld
authors.forename: Sabine
authors.initials: S
authors.lastname: Metzger
authors.forename: Magdalene
authors.initials: M
authors.lastname: Stosik
authors.forename: Norbert
authors.initials: N
authors.lastname: Tennagels
authors.forename: Jürgen
authors.initials: J
authors.lastname: Eckel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007328:Insulin / Q000737:chemistry; D007329:Insulin Antagonists / Q000737:chemistry / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D013379:Substrate Specificity; D014443:Tyrosine / Q000037:antagonists & inhibitors / Q000378:metabolism; D000099221:Protein Kinase C zeta
chemical_list: D007328:Insulin; D007329:Insulin Antagonists; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D011493:Protein Kinase C; D021122:Protein Subunits; D011972:Receptor, Insulin; D012694:Serine; D014443:Tyrosine; D000099221:Protein Kinase C zeta; C526292:IRS1 protein, human; C526296:Irs1 protein, rat