Comparison Of The Kinetic Properties Of The Lipid- And Protein-Kinase Activities Of The P110 Alpha And P110 Beta Catalytic Subunits Of Class-Ia Phosphoinositide 3-Kinases
BIOCHEMICAL JOURNAL(2000)
Abstract
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 p110 alpha and p110 beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110 alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110 beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110 beta is a better PtdIns(4,5)P(2) kinase than p110 alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110 beta functions better in areas of membranes containing low levels of substrate whereas p110 alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110 beta phosphorylated p85 to a lower degree than did p110 alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110 alpha and p110 beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110 alpha: has a higher K(m) (550 mu M) than p110 beta (K(m) 8 mu M). Similarly, the relative V(max) towards peptide substrate of p110 alpha was three times higher than that of p110 beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110 alpha: and p110 beta in vivo.
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Key words
p85 alpha, PI 3-kinase
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