Biochemical and Biophysical Research Communications
Functional differences between two classes of oncogenic mutation in the PIK3CA gene
Section snippets
Materials and methods
Materials. Unless otherwise stated other reagents were purchased from Sigma Chemicals, Auckland, NZ. PIK-75, PI-103 and polyclonal antibodies to p85 were obtained from Symansis (Auckland, NZ). Polyclonal antibodies to p110α, p110β and p110δ were kindly provided by Dr. Bart Vanhaesebroeck, Ludwig Institute, London. Polyclonal antibodies to phosphoser608 of p85α were as previously described [27]. Recombinant H-Ras (GV12) was purchased from Jena Bioscience, Jena, Germany. A doubly phosphorylated
Results and discussion
Our first finding was that both the mutant forms of p110α retain the ability to autophosphorylate p85α on Serine 608 (Fig. 1). In wild type p85α/p110α this is an autoregulatory phosphorylation [25], [27] but our results would indicate that the mutant forms of p110α are not as sensitive to this inhibitory phosphorylation.
We went on to investigate whether the mutations altered the inhibitory effects of five previously described PI 3-kinase inhibitors (Wortmannin, LY294002, PIK-75, PI-103 and SN
Acknowledgments
Funding provided by a grant from the Cancer Society of New Zealand the Maurice Wilkins Centre for Molecular Biodiscovery, the Auckland Cancer Society Research Centre and the Health Research Council of New Zealand (Project Grant 06/062A). We thank Dr. Phil Hawkins and Dr. Osamu Hazeki for providing reagents. We thank Prof. Shaun Jackson and Dr. Cenk Suphioglu for providing recombinant PI 3-kinases and Prof. Margaret Brimble for synthesizing the PDGFR-derived diphosphopeptide synthesis.
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Present address: Centre for Cell Signalling, Barts & The London School of Medicine, Queen Mary, University of London (QMUL), London.