Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.
Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.
Yonezawa
K
K
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Ueda
H
H
Hara
K
K
Nishida
K
K
Ando
A
A
Chavanieu
A
A
Matsuba
H
H
Shii
K
K
Yokono
K
K
Fukui
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000032:analysis / Q000138:chemical synthesis; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides / Q000032:analysis / Q000138:chemical synthesis; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D046911:Macromolecular Substances; D009842:Oligopeptides; D010748:Phosphopeptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D005982:Glutathione Transferase; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin