A Tightly Associated Serine/Threonine Protein Kinase Regulates Phosphoinositide 3-Kinase Activity

withantibodies raised tothe85-kDa subunit. Tryptic phosphopeptide maps indicate thatp85frompolyomavirus middleT-transformed cells is phosphorylated invivoatthree sites phosphorylated invitro bytheassociated serine kinase. The85-kDa subunit ofPtdlns 3-kinase isphosphorylated invitro on serineata stoichiometry ofapproximately 1 molof phosphate permolofp85.Thisphosphorylation results ina three- tosevenfold decrease inPtdlns 3-kinase activity. Dephosphorylation withprotein phosphatase 2A reversestheinhibition. Thissuggests thatthe association ofprotein phosphatase 2Awithmiddle T antigen may function toactivate Ptdlns 3-kinase. Inmammaliancells, phosphoinositide (PtdIns) 3-kinase associates withalmost allactivated protein-tyrosine kinases inresponsetotheappropriate stimulus (4). Association of PtdIns 3-kinase witha tyrosine-phosphorylated protein correlates withsynthesis ofphosphatidylinositol-3,4-bisphosphate(PtdIns-3,4-P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) and withcellgrowthand transformation. Studies ofcells transfected withmutantsof polyomavirus middle Tantigen (mT)(9,17,34,37), ablgene products (35), thecolony-stimulating factor 1 receptor (31), andtheplatelet-derived growthfactor (PDGF)receptor (8, 29)havedemonstrated thesecorrelations. Mostcells expressing mutantproteins thataredefective inPtdIns 3-kinase association synthesize reduced levels ofPtdIns-3,4-P2 andPtdIns-3,4,5-P3 andareimpaired intransformation and mitogenesis. Cells transfected withmutantsofabl(35) and mT (20)thatassociate withPtdIns 3-kinase butdo not transform havereduced levels ofPtdIns-3,4-P2 andPtdIns3,4,5-P3 compared withtransformed cells. Thelevel ofthese lipids isa better predictor ofcell transformation thanisthe association ofPtdIns 3-kinase withoncoproteins. PtdIns 3-kinase hasbeenpurified fromratliver (5), bovine brain(23,24), mouse fibroblasts (12), andbovinethymus (30). Theenzyme purified fromratliver isa heterodimer of an 85-kDaandone oftwo110-kDa proteins. Itphosphorylates theD-3hydroxyl ofphosphoinositides toproduce phosphatidylinositol-3-phosphate (PtdIns-3-P), PtdIns-3,4P2,andPtdIns-3,4,5-P3 (2,5,36). ThecDNA encoding the 85-kDasubunit ofPtdIns 3-kinase hasbeencloned (12, 24, 32).Two distinct cDNAshavebeenobtained frombovine brain, 85aand851(24). Bothclones encodeproteins with twoSH2domainsandan SH3domain.Bothproteins also contain adomainhomologous tothecarboxy terminus ofthe breakpoint cluster region gene product andtheGTPaseactivating proteins forthesmallGTP binding proteins rho andrac (10).