Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110alpha and p110beta catalytic subunits of class-Ia phosphoinositide 3-kinases.
Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110alpha and p110beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110beta is a better PtdIns(4,5)P(2) kinase than p110alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110beta functions better in areas of membranes containing low levels of substrate whereas p110alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110beta phosphorylated p85 to a lower degree than did p110alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110alpha and p110beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110alpha has a higher K(m) (550 microM) than p110beta (K(m) 8 microgM). Similarly, the relative V(max) towards peptide substrate of p110alpha was three times higher than that of p110beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110alpha and p110beta in vivo.
Beeton
C A
CA
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
Chance
E M
EM
Foukas
L C
LC
Shepherd
P R
PR
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011233:Precipitin Tests; D020033:Protein Isoforms; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C068961:phosphatidylinositol 4,5-biphosphate kinase; D000077191:Wortmannin