Phosphorylation of the insulin receptor substrate IRS-1 by casein kinase II.
IRS-1, a principal substrate of the insulin receptor, is phosphorylated on serine, threonine, and tyrosine residues in a variety of tissues during insulin stimulation. Casein kinase II, an insulin-sensitive serine/threonine kinase, catalyzed the in vitro incorporation of 1 to 2 mol of phosphate/mol of recombinant rat IRS-1. Two-dimensional phosphopeptide mapping of IRS-1 phosphorylated by casein kinase II in vitro and IRS-1 immunoprecipitated from intact Chinese hamster ovary cells demonstrated multiple common phosphopeptides, suggesting that overexpressed IRS-1 is a substrate for casein kinase II in these cells. Moreover, the common phosphopeptides that appeared to be insulin-sensitive in intact cells comprised 22% of casein kinase II-catalyzed 32P incorporation into IRS-1 in vitro. These data suggest that casein kinase II mediates a portion of the insulin-stimulated serine/threonine phosphorylation of overexpressed IRS-1 in vivo. By using phosphoamino acid analysis, strong cation exchange analysis, manual Edman degradation, and automated amino acid sequencing, Thr-502 was identified as the major casein kinase II-catalyzed phosphorylation site in rat IRS-1. Furthermore, Ser-99, an additional site labeled at low yield, appeared to be contained in an insulin-sensitive phosphopeptide. Thus, casein kinase II-catalyzed phosphorylation of IRS-1 may be a component of the intracellular insulin signalling cascade.
Tanasijevic
M J
MJ
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Myers
M G
MG
Thoma
R S
RS
Crimmins
D L
DL
White
M F
MF
Sacks
D B
DB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D016466:CHO Cells; D047390:Casein Kinase II; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D000596:Amino Acids; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010748:Phosphopeptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D011972:Receptor, Insulin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases