title: The p85 and p110 subunits of phosphatidylinositol 3-kinase-alpha are substrates, in vitro, for a constitutively associated protein tyrosine kinase in platelets.
abstract: Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn(2+)-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn(2+)-dependent protein kinase activity of PI3K alpha immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3K alpha showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85 alpha or p110 alpha derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85 alpha and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3K alpha can autophosphorylate on serine and threonine, and both p85 alpha and p110 alpha are substrates for a constitutively-associated protein tyrosine kinase in platelets.
authors.affiliation: Blood Research Institute, Milwaukee, WI 53226-3548, USA.
authors.forename: N R
authors.initials: NR
authors.lastname: Geltz
authors.forename: J A
authors.initials: JA
authors.lastname: Augustine
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002867:Chromones / Q000494:pharmacology; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009025:Morpholines / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000097:blood*; D013379:Substrate Specificity; D013917:Thrombin / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D009842:Oligopeptides; C096900:thrombin receptor-activating peptide SFLLRNPNDKY; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D008345:Manganese; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D013917:Thrombin; D000077191:Wortmannin