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 p110α and p110β isoforms. The lipid-kinase activity did not display Michaelis–Menten kinetics but modelling the kinetic data demonstrated that p110α has a higher Vmax and a 25-fold higher Km for PtdIns than p110β. A similar situation occurs with PtdIns(4,5)P2, because at low concentration of PtdIns(4,5)P2 p110β is a better PtdIns(4,5)P2 kinase than p110α, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110β functions better in areas of membranes containing low levels of substrate whereas p110α would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110β phosphorylated p85 to a lower degree than did p110α. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110α and p110β. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110α has a higher Km (550µM) than p110β (Km 8µM). Similarly, the relative Vmax towards peptide substrate of p110α was three times higher than that of p110β. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110α and p110β in vivo.
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Research Article|
August 23 2000
Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110α and p110β catalytic subunits of class-Ia phosphoinositide 3-kinases
Carolyn A. BEETON;
Carolyn A. BEETON
1Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
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Edwin M. CHANCE;
Edwin M. CHANCE
1Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
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Lazaros C. FOUKAS;
Lazaros C. FOUKAS
1Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
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Peter R. SHEPHERD
Peter R. SHEPHERD
1
1Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
1To whom correspondence should be addressed (e-mail p.shepherd@biochem.ucl.ac.uk).
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Publisher: Portland Press Ltd
Received:
March 31 2000
Revision Received:
June 12 2000
Accepted:
July 06 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 350 (2): 353–359.
Article history
Received:
March 31 2000
Revision Received:
June 12 2000
Accepted:
July 06 2000
Citation
Carolyn A. BEETON, Edwin M. CHANCE, Lazaros C. FOUKAS, Peter R. SHEPHERD; Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110α and p110β catalytic subunits of class-Ia phosphoinositide 3-kinases. Biochem J 1 September 2000; 350 (2): 353–359. doi: https://doi.org/10.1042/bj3500353
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