PHOSPHORYLATION OF THE INSULIN-RECEPTOR SUBSTRATE IRS-1 BY CASEIN KINASE-II

IRS-1, a principal substrate of the insulin receptor, is phosphorylate d on serine, threonine, and tyrosine residues in a variety of tissues during insulin stimulation. Casein kinase II, an insulin-sensitive ser ine/threonine kinase, catalyzed the in vitro incorporation of 1 to 2 m ol of phosphate/mol of recombinant rat IRS-1. Two-dimensional phosphop eptide mapping of IRS-1 phosphorylated by casein kinase II in vitro an d IRS-1 immunoprecipitated from intact Chinese hamster ovary cells dem onstrated multiple common phosphopeptides, suggesting that overexpress ed IRS-1 is a substrate for casein kinase II in these cells. Moreover, the common phosphopeptides that appeared to be insulin-sensitive in i ntact cells comprised 22% of casein kinase II-catalyzed P-32 incorpora tion into IRS-1 in vitro. These data suggest that casein kinase II med iates a portion of the insulin-stimulated serine/threonine phosphoryla tion of overexpressed IRS-1 in vivo. By using phosphoamino acid analys is, strong cation exchange analysis, manual Edman degradation, and aut omated amino acid sequencing, Thr-502 was identified as the major case in kinase II-catalyzed phosphorylation site in rat IRS-1. Furthermore, Ser-99, an additional site labeled at low yield, appeared to be conta ined in an insulin-sensitive phosphopeptide. Thus, casein kinase II-ca talyzed phosphorylation of IRS-1 may be a component of the intracellul ar insulin signalling cascade.