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Regulation of lipid binding underlies the activation mechanism of class IA PI3-kinases.

ABSTRACT: Somatic missense mutations in PIK3CA, which encodes the p110? catalytic subunit of phosphoinositide 3-kinases, occur frequently in human cancers. Activating mutations spread across multiple domains, some of which are located at inhibitory contact sites formed with the regulatory subunit p85?. PIK3R1, which encodes p85?, also has activating somatic mutations. We find a strong correlation between lipid kinase and lipid-binding activities for both wild-type (WT) and a representative set of oncogenic mutant complexes of p110?/p85?. Lipid binding involves both electrostatic and hydrophobic interactions. Activation caused by a phosphorylated receptor tyrosine kinase (RTK) peptide binding to the p85? N-terminal SH2 domain (nSH2) induces lipid binding. This depends on the polybasic activation loop as well as a conserved hydrophobic motif in the C-terminal region of the kinase domain. The hotspot E545K mutant largely mimics the activated WT p110?. It shows the highest basal activity and lipid binding, and is not significantly activated by an RTK phosphopeptide. Both the hotspot H1047R mutant and rare mutations (C420R, M1043I, H1047L, G1049R and p85?-N564D) also show increased basal kinase activities and lipid binding. However, their activities are further enhanced by an RTK phosphopeptide to levels markedly exceeding that of activated WT p110?. Phosphopeptide binding to p110?/p85? and p110?/p85? complexes also induces their lipid binding. We present a crystal structure of WT p110? complexed with the p85? inter-SH2 domain and the inhibitor PIK-108. Additional to the ATP-binding pocket, an unexpected, second PIK-108 binding site is observed in the kinase C-lobe. We show a global conformational change in p110? consistent with allosteric regulation of the kinase domain by nSH2. These findings broaden our understanding of the differential biological outputs exhibited by distinct types of mutations regarding growth factor dependence, and suggest a two-tier classification scheme relating p110? and p85? mutations with signalling potential.

SUBMITTER: Hon WC

PROVIDER: S-EPMC3378484 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Regulation of lipid binding underlies the activation mechanism of class IA PI3-kinases.

Hon W-C WC Berndt A A Williams R L RL

Oncogene 20111128 32

Somatic missense mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide 3-kinases, occur frequently in human cancers. Activating mutations spread across multiple domains, some of which are located at inhibitory contact sites formed with the regulatory subunit p85α. PIK3R1, which encodes p85α, also has activating somatic mutations. We find a strong correlation between lipid kinase and lipid-binding activities for both wild-type (WT) and a representative set of oncogeni ...[more]

PMID: 22120714

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Project description:We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (?, ?, ? and ?) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110?, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110?>>?>? with undetectable p110?. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110?-, but not ?- or ?- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110?, but not ?- or ?- activity. In the presence of single, endogenous alleles of onco-mutant p110? (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110? inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110?, but not ?- or ?- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110?-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110? is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110? augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110? function in the absence of PTEN.

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Regulation of lipid binding underlies the activation mechanism of class IA PI3-kinases.

Publications

Regulation of lipid binding underlies the activation mechanism of class IA PI3-kinases.

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