Synergistic activation of PtdIns 3-kinase by tyrosine-phosphorylated peptide and βγ-subunits of GTP-binding proteins

Stimulation of differentiated THP-1 cells by insulin led to rapid accumulation of PtdIns(3,4,5)P $ , a product of PtdIns 3-kinase. Stimulation of the GTP-binding-protein-linked receptor by Nformylmethionyl-leucyl-phenylalanine (fMLP) also induced the accumulation of PtdIns(3,4,5)P $ 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 immunoprecipitate. The combination of insulin and fMLP induced more PtdIns(3,4,5)P $ 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)P $ , we separated the cytosolic proteins of THP-1 cells on a Mono Q