Protein kinase C-zeta-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.
Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. 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-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, 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-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-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-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.
Moeschel
Klaus
K
Department of Internal Medicine IV, Endocrinology, Metabolism, Pathobiochemistry and Clinical Chemistry, University Hospital Tübingen, D-72076 Tübingen, Germany.
Beck
Alexander
A
Weigert
Cora
C
Lammers
Reiner
R
Kalbacher
Hubert
H
Voelter
Wolfgang
W
Schleicher
Erwin D
ED
Häring
Hans-Ulrich
HU
Lehmann
Rainer
R
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D018832:Molecular Chaperones / Q000502:physiology*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D000099221:Protein Kinase C zeta
D048948:14-3-3 Proteins; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D018832:Molecular Chaperones; D010750:Phosphoproteins; D011972:Receptor, Insulin; D012694:Serine; D014443:Tyrosine; C526292:IRS1 protein, human; C526296:Irs1 protein, rat; C490392:Prkcz protein, rat; D000099221:Protein Kinase C zeta; C490391:Ywhaz protein, rat; C090759:mitochondrial import stimulation factor, rat