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Dynamic steps in receptor tyrosine kinase mediated activation of class IA phosphoinositide 3-kinases (PI3K) captured by H/D exchange (HDX-MS).

ABSTRACT: The catalytic subunits of all class IA phosphoinositide 3-kinases (PI3Ks) associate with identical p85-related subunits and phosphorylate PIP2 yielding PIP3, but they can vary greatly in the signaling pathways in which they participate. The binding of the p85 subunit to the p110 catalytic subunits is constitutive, and this inhibits activity, but some of the inhibitory contacts are reversible and subject to regulation. Interaction with phosphotyrosine-containing peptides (RTK-pY) releases a subset of these inhibitory contacts. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) provides a map of the dynamic interactions unique to each of the isotypes. RTK-pY binding exposes the p110 helical domains for all class IA enzymes (due to release of the nSH2 contact) and exposes the C-lobe of the kinase domains of p110? and p110? (resulting from release of the cSH2 contact). Consistent with this, our in vitro assays show that all class IA isoforms are inhibited by the nSH2, but only p110? and p110? are inhibited by the cSH2. While a C2/iSH2 inhibitory contact exists in all isoforms, HDX indicates that p110? releases this contact most readily. The unique dynamic relationships of the different p110 isozymes to the p85 subunit may facilitate new strategies for specific inhibitors of the PI3Ks.

SUBMITTER: Burke JE

PROVIDER: S-EPMC3613897 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Dynamic steps in receptor tyrosine kinase mediated activation of class IA phosphoinositide 3-kinases (PI3K) captured by H/D exchange (HDX-MS).

Burke John E JE Williams Roger L RL

Advances in biological regulation 20120913 1

The catalytic subunits of all class IA phosphoinositide 3-kinases (PI3Ks) associate with identical p85-related subunits and phosphorylate PIP2 yielding PIP3, but they can vary greatly in the signaling pathways in which they participate. The binding of the p85 subunit to the p110 catalytic subunits is constitutive, and this inhibits activity, but some of the inhibitory contacts are reversible and subject to regulation. Interaction with phosphotyrosine-containing peptides (RTK-pY) releases a subse ...[more]

PMID: 23194976

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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.

Dataset Information

Dynamic steps in receptor tyrosine kinase mediated activation of class IA phosphoinositide 3-kinases (PI3K) captured by H/D exchange (HDX-MS).

Publications

Dynamic steps in receptor tyrosine kinase mediated activation of class IA phosphoinositide 3-kinases (PI3K) captured by H/D exchange (HDX-MS).

Similar Datasets

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