Purification and characterization of Gβγ-responsive phosphoinositide 3-kinases from pig platelet cytosol

Abstract

A G-protein βγ subunit (Gβγ)-responsive phosphoinositide 3-kinase (PI 3-kinase) was purified approximately 5000-fold from pig platelet cytosol. The enzyme was purified by polyethylene glycol precipitation of the cytosol followed by column chromatography on Q-Sepharose fast flow, gel filtration, heparin-Sepharose, and hydroxyapatite. The major Gβγ-responsive PI 3- kinase is distinct from p85 containing PI 3-kinase as the activities can be distinguished chromatographically and immunologically and is related to p110γ as it crossreacts with anti-p110γ-specific antibodies. The p110γ- related PI 3-kinase cannot be activated by G-protein α(i/o) subunits, and it has an apparent native molecular mass of 210 kDa. The p110γ-related PI 3- kinase phosphorylates phosphatidylinositol (PtdIns), phosphatidylinositol 4- phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The apparent K(m) values for ATP were found to be 25 μM with PtdIns, 44 μM with PtdIns4P, and 37 μM with PtdIns(4,5)P2 as the substrate. Gβγ subunits did not alter the K(m) of the enzyme for ATP; however, V(max) increased 2-fold with PtdIns as substrate, 3.5-fold with PtdIns4P, and 10-fold with PtdIns(4,5)P2. Under basal conditions the apparent K(m) values for lipid substrates were 64, 10, and 15 μM for PtdIns, PtdIns4P, and PtdIns(4,5)P2, respectively. In the presence of Gβγ subunits the dependence of PI 3-kinase activity on the concentrations of lipid substrates became complex with the highest level of stimulation occurring at high substrate concentration, suggesting that the binding of Gβγ and lipid substrate (particularly PtdIns(4,5)P2) may be mutually cooperative. Wortmannin and LY294002 inhibit the Gβγ-responsive PI 3-kinase activity with IC50 values of 10 nM and 2 μM, respectively. Unlike the p85 containing PI 3-kinase in platelets, the p110γ-related PI 3-kinase is not associated with a PtdIns(3,4,5)P3 specific 5-phosphatase. The p85-associated PI 3-kinase was not activated by Gβγ alone but could be synergistically activated by Gβγ and phosphotyrosyl platelet-derived growth factor receptor peptides. This may represent a form of coincidence detection through which the effects of tyrosine kinase and G-protein-linked receptors might be coordinated.