Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCδ and PKCθ 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, PKCδ, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCδ or wild-type PKCδ followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCδ 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 PKCδ acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCδ on at least 18 sites. The importance of three of the PKCδ 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 PKCδ on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCδ modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.
- insulin resistance
- IRS-1 (insulin receptor substrate-1)
- nPKC (novel protein kinase C)
- tyrosine phosphorylation
Abbreviations used: CHO/IR cells, Chinese-hamster ovary cells overexpressing the insulin receptor; DAG, diacylglycerol; DTT, dithiothreitol; GST, glutathione S-transferase; hIR, human insulin receptor; IRS, insulin receptor substrate; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; MAPK, mitogen-activated protein kinase; NEFA, non-esterified fatty acid (free fatty acid); PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; aPKC, cPKC and nPKC, atypical, classical and novel PKC respectively; PS, phosphatidylserine; PTB domain, phosphotyrosine binding domain; SH2, Src homology 2; wt, wild-type.
- The Biochemical Society, London ©2004