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Regulation of Insulin Receptor Substrate 1 Pleckstrin Homology Domain by Protein Kinase C: Role of Serine 24 Phosphorylation

Department of Clinical Biochemistry (R.N., C.H.J., K.S., J.K.S.), University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QR, United Kingdom; and School of Life Sciences (A.G., C.P.D.), University of Dundee, Dundee DD1 5EH, United Kingdom

Address all correspondence and requests for reprints to: Jaswinder K. Sethi, Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Box 232, Hills Road, Cambridge CB2 2QR, United Kingdom. E-mail: jks30{at}cam.ac.uk.

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKC. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKC is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.

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