PURIFICATION AND CHARACTERIZATION OF G-BETA-GAMMA-RESPONSIVE PHOSPHOINOSITIDE 3-KINASES FROM PIG PLATELET CYTOSOL

A G-protein beta gamma subunit (G beta gamma)-responsive phosphoinosit ide 3-kinase (PI 3-kinase) was purified approximately 5000-fold from p ig platelet cytosol, The enzyme was purified by polyethylene glycol pr ecipitation of the cytosol followed by column chromatography on Q-Seph arose fast flow, gel filtration, heparin-Sepharose, and hydroxyapatite . The major G beta gamma-responsive PI 3-kinase is distinct from p85 c ontaining PI 3-kinase as the activities can be distinguished chromatog raphically and immunologically and is related to p110 gamma as it cros sreacts with anti-p110 gamma-specific antibodies. The p110 gamma-relat ed PI 3-kinase cannot be activated by G-protein alpha(i/o) subunits, a nd it has an apparent native molecular mass of 210 kDa, The p110 gamma -related PI 3-kinase phosphorylates phosphatidylinositol (PtdIns), pho sphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5 -bisphosphate (PtdIns(4,5)P-2). The apparent K-m values for ATP were f ound to be 25 mu M with PtdIns, 44 mu M with PtdIns4P, and 37 mu M wit h PtdIns(4,5)P-2 as the substrate, G beta gamma subunits did not alter the K-m of the enzyme for ATP; however, V-max increased 2-fold with P tdIns as substrate, 3.5-fold with PtdIns4P, and 10-fold with PtdIns(4, 5)P-2. Under basal conditions the apparent K-m values for lipid substr ates were 64, 10, and 15 mu M for PtdIns, PtdIns4P, and PtdIns(4,5)P-2 , respectively, In the presence of G beta gamma subunits the dependenc e of PI 3-kinase activity on the concentrations of lipid substrates be came complex with the highest level of stimulation occurring at high s ubstrate concentration, suggesting that the binding of G beta gamma an d lipid substrate (particularly PtdIns(4,5)P-2) may be mutually cooper ative, Wortmannin and LY294002 inhibit the G beta gamma-responsive PI 3-kinase activity with IC50 values of 10 nM and 2 mu M, respectively, Unlike the p85 containing PI 3-kinase in platelets, the p110 gamma-rel ated PI 3-kinase is not associated with a PtdIns(3,4,5)P-3 specific 5- phosphatase. The p85-associated PI 3-kinase was not activated by G bet a gamma alone but could be synergistically activated by G beta gamma a nd phosphotyrosyl platelet-derived growth factor receptor peptides, Th is may represent a form of coincidence detection through which the eff ects of tyrosine kinase and G-protein-linked receptors might be coordi nated.