Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta.
Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCdelta and PKCtheta correlates with insulin resistance, including decreased insulin-stimulated IRS-1 (insulin receptor substrate-1) tyrosine phosphorylation and phosphoinositide 3-kinase activation, although the mechanism(s) for this resistance is not known. In the present study, we have explored the possibility of a novel PKC, PKCdelta, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCdelta or wild-type PKCdelta followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCdelta was also found to inhibit the IRS-1 tyrosine phosphorylation in vitro by purified insulin receptor using recombinant full-length human IRS-1 and a partial IRS-1-glutathione S-transferase-fusion protein as substrates. This inhibition in vitro was not observed with a non-IRS-1 substrate, indicating that it was not the result of a general decrease in the intrinsic kinase activity of the receptor. Consistent with the hypothesis that PKCdelta acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCdelta on at least 18 sites. The importance of three of the PKCdelta phosphorylation sites in IRS-1 was shown in vitro by a 75-80% decrease in the incorporation of phosphate into an IRS-1 triple mutant in which Ser-307, Ser-323 and Ser-574 were replaced by Ala. More importantly, the mutation of these three sites completely abrogated the inhibitory effect of PKCdelta on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCdelta modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Michael W
MW
Greene
Nick
N
Morrice
Robert S
RS
Garofalo
Richard A
RA
Roth
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D010455:Peptides; D010750:Phosphoproteins; D014443:Tyrosine; C410467:insulin receptor tyrosine kinase; D011505:Protein-Tyrosine Kinases; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta