Synergistic activation of PtdIns 3-kinase by tyrosine-phosphorylated peptide and beta gamma-subunits of GTP-binding proteins.
Stimulation of differentiated THP-1 cells by insulin led to rapid accumulation of PtdIns(3,4,5)P3, a product of PtdIns 3-kinase. Stimulation of the GTP-binding-protein-linked receptor by N-formylmethionyl-leucyl-phenylalanine (fMLP) also induced the accumulation of PtdIns(3,4,5)P3 in the cells. The effect of insulin was, while that of fMLP was not, accompanied by increased PtdIns 3-kinase activity in the anti-phosphotyrosine immuno-precipitate. The combination of insulin and fMLP induced more PtdIns(3,4,5)P3 production than the sum of the individual effects. The insulin-induced recruitment of PtdIns 3-kinase activity in the anti-phosphotyrosine immunoprecipitate was unaffected by the combined treatment with fMLP. To investigate the mechanism underlying the synergistic accumulation of PtdIns(3,4,5)P3, we separated the cytosolic proteins of THP-1 cells on a Mono Q column. PtdIns 3-kinase activities were eluted in two peaks, and one of the peaks markedly increased on the addition of beta gamma-subunits of GTP-binding proteins (G beta gamma). The other peak was affected only slightly by G beta gamma, but was synergistically increased by G beta gamma and a tyrosine-phosphorylated peptide which was synthesized accordingly to the amino acid sequence of insulin receptor substrate-1. The activity in the latter fraction was completely immunoprecipitated by an antibody against the regulatory subunit of PtdIns 3-kinase (p85). These results suggest that the conventional PtdIns 3-kinase (p85/p110), which has been implicated in insulin-induced cellular events, or a closely related isoenzyme is controlled by a combination of a tyrosine-phosphorylated protein and a GTP-binding protein in intact cells.
Okada
T
T
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Hazeki
O
O
Ui
M
M
Katada
T
T
D000595:Amino Acid Sequence; D002474:Cell-Free System / Q000378:metabolism; D004357:Drug Synergism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010748:Phosphopeptides / Q000494:pharmacology*; D010750:Phosphoproteins / Q000494:pharmacology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D015398:Signal Transduction
C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D010748:Phosphopeptides; D010750:Phosphoproteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019204:GTP-Binding Proteins