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

Abstract

Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-ζ. 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-ζ phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-ζ, we identified Ser318 of rat IRS-1 (Ser323 in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser318 to alanine). To monitor phosphorylation of Ser318 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 (BHKIR). Using the phospho-Ser318-specific antibody we observed that insulin stimulates phosphorylation of Ser318 in IRS-1, which is mediated, at least partially, by PKC-ζ. Moreover, we found that the previously described insulin-stimulated, PKC-ζ-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 Ser318 to alanine. These results, generated in BHKIR cells, suggest that phosphorylation of Ser318 by PKC-ζ might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function. Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-ζ. 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-ζ phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-ζ, we identified Ser318 of rat IRS-1 (Ser323 in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser318 to alanine). To monitor phosphorylation of Ser318 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 (BHKIR). Using the phospho-Ser318-specific antibody we observed that insulin stimulates phosphorylation of Ser318 in IRS-1, which is mediated, at least partially, by PKC-ζ. Moreover, we found that the previously described insulin-stimulated, PKC-ζ-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 Ser318 to alanine. These results, generated in BHKIR cells, suggest that phosphorylation of Ser318 by PKC-ζ might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.