Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

Meredith J Layton, Natalie K Rynkiewicz, Ivan Ivetac, Kristy A Horan, Christina Anne Mitchell, Wayne A Phillips

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Oncogenic mutations in PIK3CA lead to an increase in instrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kalpha to its plasma membrane localised substrate is also required for increased levels of downstream PIP3/Akt signalling. We have studied the subcellular localisation of wild type and the two most common oncogenic mutants of PI3Kalpha in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kalpha is pre-formed as a 1:1 p110alpha:p85alpha complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipid in vitro or in cells maintained in 10 FCS. Following stimulation of receptor tyrosine kinases, microinjected PI3Kalpha was recruited to the plasma membrane, but oncogenic forms of PI3Kalpha were not recruited to the plasma membrane to a greater extent and did not reside at the plasma membrane longer than wild type PI3Kalpha. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein-protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.
Original languageEnglish
Pages (from-to)165 - 180
Number of pages16
JournalBioscience Reports
Volume34
Issue number2 (Art. ID: e00104)
DOIs
Publication statusPublished - 2014

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