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. 1996 Jul 15;317(Pt 2):475–480. doi: 10.1042/bj3170475

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

T Okada 1, O Hazeki 1, M Ui 1, T Katada 1
PMCID: PMC1217511  PMID: 8713074

Abstract

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.

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Selected References

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