Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation.
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 PKCalpha. 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 PKCalpha 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.
Nawaratne
Ranmali
R
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, UK.
Gray
Alexander
A
Jørgensen
Christina H
CH
Downes
C Peter
CP
Siddle
Kenneth
K
Sethi
Jaswinder K
JK
D041721:3T3-L1 Cells; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D020134:Catalytic Domain; D002518:Ceramides / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D051571:Protein Kinase C-alpha / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D001798:Blood Proteins; D002518:Ceramides; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; C039077:platelet protein P47; D012694:Serine; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate