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Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta

Michael W Greene1, Nick Morrice, Robert S Garofalo

  • 1Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA.

The Biochemical Journal
|October 30, 2003
PubMed

Insights

Protein kinase C delta (PKCdelta) directly phosphorylates insulin receptor substrate-1 (IRS-1), inhibiting insulin signaling. This finding reveals a novel mechanism linking PKCdelta activation to insulin resistance.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Signaling

Background:

  • Non-esterified fatty acid-induced activation of novel protein kinase C (PKC) isoenzymes PKCdelta and PKCtheta is linked to insulin resistance.
  • This resistance involves decreased insulin-stimulated IRS-1 tyrosine phosphorylation and phosphoinositide 3-kinase activation, but the underlying mechanisms remain unclear.

Purpose of the Study:

  • To investigate whether PKCdelta directly modulates the insulin receptor kinase's ability to tyrosine-phosphorylate IRS-1.
  • To elucidate the specific molecular mechanisms by which PKCdelta influences insulin signaling.

Main Methods:

  • Expression of constitutively active or activated wild-type PKCdelta in cells to assess its effect on insulin-stimulated IRS-1 tyrosine phosphorylation in vivo.
  • In vitro kinase assays using purified insulin receptor, recombinant IRS-1, and a glutathione S-transferase-IRS-1 fusion protein to evaluate direct inhibition by activated PKCdelta.
  • Site-directed mutagenesis of IRS-1 to identify key phosphorylation sites targeted by PKCdelta and assess their impact on tyrosine phosphorylation.

Main Results:

  • Expression of activated PKCdelta inhibited insulin-stimulated IRS-1 tyrosine phosphorylation both in vivo and in vitro.
  • Activated PKCdelta inhibited IRS-1 tyrosine phosphorylation in vitro without affecting a non-IRS-1 substrate, indicating specificity.
  • PKCdelta directly phosphorylated IRS-1 on at least 18 sites, with mutations at Ser-307, Ser-323, and Ser-574 significantly reducing phosphate incorporation and abrogating the inhibitory effect of PKCdelta on IRS-1 tyrosine phosphorylation.

Conclusions:

  • PKCdelta directly modulates the insulin receptor's ability to tyrosine-phosphorylate IRS-1.
  • Direct phosphorylation of IRS-1 by PKCdelta at specific sites (Ser-307, Ser-323, Ser-574) is a key mechanism underlying PKCdelta-induced inhibition of insulin signaling and contributes to insulin resistance.

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