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PKCdelta-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

Michael W Greene1, Mary S Ruhoff, Richard A Roth

  • 1Bassett Research Institute, Mary Imogene Bassett Hospital, Cooperstown, NY 13326, USA. michael.greene@bassett.org <michael.greene@bassett.org>

Biochemical and Biophysical Research Communications
|September 15, 2006
PubMed

Insights

Insulin receptor substrate-1 (IRS-1) Ser24 phosphorylation by PKCdelta acts as a negative regulator, inhibiting insulin signaling and cellular proliferation by impairing phosphatidylinositol-4,5-bisphosphate binding.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • Insulin receptor substrate-1 (IRS-1) is crucial for insulin signaling, with its tyrosine phosphorylation regulated by both positive and negative mechanisms.
  • Serine/threonine phosphorylation of IRS-1, particularly by Protein Kinase C (PKC), can disrupt insulin signaling pathways.

Purpose of the Study:

  • To investigate the role of specific serine/threonine phosphorylation sites within the PH-PTB domains of human IRS-1.
  • To determine if consensus PKC phosphorylation sites (Ser24, Ser58, Thr191) are involved in the negative regulation of insulin signaling.

Main Methods:

  • Site-directed mutagenesis was used to create IRS-1 mutants where Ser24, Ser58, and Thr191 were replaced with Alanine (to block phosphorylation) or Glutamate (to mimic phosphorylation).
  • These mutants were analyzed for their effects on insulin-stimulated IRS-1 tyrosine phosphorylation, cellular proliferation, and Akt activation.
  • PKCdelta involvement was confirmed using catalytic domain mutants and RNA interference (RNAi).
  • Phosphatidylinositol-4,5-bisphosphate binding assays were performed on mutated IRS-1 proteins.

Main Results:

  • Mutating Ser24, Ser58, and Thr191 to Alanine (3A) abrogated the inhibitory effect of PKCdelta on IRS-1 tyrosine phosphorylation.
  • Mutating these sites to Glutamate (3E) reduced insulin-stimulated IRS-1 tyrosine phosphorylation, cellular proliferation, and Akt activation.
  • The Ser24 to Glutamate single mutant showed the most significant inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation.
  • PKCdelta-mediated phosphorylation at Ser24 was confirmed, and mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding.

Conclusions:

  • Serine 24 in IRS-1 is identified as a key negative regulatory phosphorylation site.
  • PKCdelta-mediated phosphorylation of IRS-1 at Ser24 inhibits insulin signaling by disrupting phosphatidylinositol-4,5-bisphosphate binding.
  • This finding provides mechanistic insight into how IRS-1 activity is downregulated in response to certain stimuli.

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