Purification and characterization of Gbetagamma-responsive phosphoinositide 3-kinases from pig platelet cytosol.
A G-protein betagamma subunit (Gbetagamma)-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 Gbetagamma-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 p110gamma as it cross-reacts with anti-p110gamma-specific antibodies. The p110gamma-related PI 3-kinase cannot be activated by G-protein alphai/o subunits, and it has an apparent native molecular mass of 210 kDa. The p110gamma-related PI 3-kinase phosphorylates phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The apparent Km values for ATP were found to be 25 microM with PtdIns, 44 microM with PtdIns4P, and 37 microM with PtdIns(4,5)P2 as the substrate. Gbetagamma subunits did not alter the Km of the enzyme for ATP; however, Vmax 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 Km values for lipid substrates were 64, 10, and 15 microM for PtdIns, PtdIns4P, and PtdIns(4,5)P2, respectively. In the presence of Gbetagamma 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 Gbetagamma and lipid substrate (particularly PtdIns(4,5)P2) may be mutually cooperative. Wortmannin and LY294002 inhibit the Gbetagamma-responsive PI 3-kinase activity with IC50 values of 10 nM and 2 microM, respectively. Unlike the p85 containing PI 3-kinase in platelets, the p110gamma-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 Gbetagamma alone but could be synergistically activated by Gbetagamma 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.
Tang
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Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland.
Downes
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D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology* / Q000648:ultrastructure; D002867:Chromones / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019204:GTP-Binding Proteins; D000077191:Wortmannin