In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.
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.
Sommerfeld
Mark R
MR
Department of Clinical Biochemistry and Pathobiochemistry, German Diabetes Research Institute, Germany.
Metzger
Sabine
S
Stosik
Magdalene
M
Tennagels
Norbert
N
Eckel
Jürgen
J
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
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