Functional differences between two classes of oncogenic mutation in the PIK3CA gene

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Abstract

PIK3CA codes for the p110α isoform of class-IA PI 3-kinase and oncogenic mutations in the helical domain and kinase domain are common in several cancers. We studied the biochemical properties of a common helical domain mutant (E545K) and a common kinase domain mutant (H1047R). Both retain the ability to autophosphorylate Ser608 of p85α and are also inhibited by a range of PI 3-kinase inhibitors (Wortmannin, LY294002, PI-103 and PIK-75) to a similar extent as WT p110α. Both mutants display an increased Vmax but while a PDGF derived diphosphotyrosylpeptide caused an increase in Vmax for WT p85α/p110α it did not for the E545K variant and actually decreased Vmax for the H1047R variant. Further, the E545K mutant was activated by H-Ras whereas the H1047R mutant was not. Together these results suggest helical domain mutants are in a state mimicking activation by growth factors whereas kinase domain mutants mimic the state activated by H-Ras.

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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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      E545K and H1047R are the most frequent mutations occurring in PI3Kα helical and kinase domain respectively. Although the exact molecular mechanism by which these mutations alter PI3Kα enzymatic activity is not completely understood, several evidences suggest that the introduction of E545K into the helical domain determines the loss of the p85 inhibitory activity on p110α catalytic subunit (Chaussade et al., 2009). In a physiologic context, the p85 subunit blocks the interaction of p110α with PtdIns substrates and ATP.

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    Present address: Centre for Cell Signalling, Barts & The London School of Medicine, Queen Mary, University of London (QMUL), London.

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