Protein Kinase C-ζ Phosphorylates Insulin Receptor Substrate-1 and Impairs Its Ability to Activate Phosphatidylinositol 3-Kinase in Response to Insulin

• Published in: The Journal of biological chemistry 2001-02, Vol.276 (5), p.3543-3549
• Main Authors: Ravichandran, Lingamanaidu V., Esposito, Diana L., Chen, Judy, Quon, Michael J.
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Animals; Enzyme Activation; Humans; Insulin - physiology; Insulin Receptor Substrate Proteins; Mice; Phosphatidylinositol 3-Kinases - metabolism; Phosphoproteins - metabolism; Phosphorylation; Protein Kinase C - metabolism; Signal Transduction - physiology; Substrate Specificity; Tyrosine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M007231200

Protein kinase C-ζ (PKC-ζ) is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-ζ that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-ζ. Under in vitro conditions, wild-type PKC-ζ (but not kinase-deficient mutant PKC-ζ) significantly phosphorylated IRS-1. This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A. In addition, the overexpression of PKC-ζ in NIH-3T3IR cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [32P]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-ζ, and this association was increased 2-fold upon insulin stimulation. Furthermore, the overexpression of PKC-ζ in NIH-3T3IRcells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-1-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-ζ. Because PKC-ζ is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-ζ may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.