title: Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells.
abstract: Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, VIC 3800, Australia.
authors.forename: Meredith J
authors.initials: MJ
authors.lastname: Layton
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, VIC 3800, Australia.
authors.forename: Natalie K
authors.initials: NK
authors.lastname: Rynkiewicz
authors.affiliation: Surgical Oncology Research Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC 3002, Australia.
authors.forename: Ivan
authors.initials: I
authors.lastname: Ivetac
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, VIC 3800, Australia.
authors.forename: Kristy A
authors.initials: KA
authors.lastname: Horan
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, VIC 3800, Australia.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Mitchell
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, VIC 3800, Australia wayne.phillips@petermac.org.|Surgical Oncology Research Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC 3002, Australia.|Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia.|Department of Surgery (St. Vincent's Hospital), University of Melbourne, Parkville, VIC 3010, Australia.
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Phillips
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D058534:Class I Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008563:Membrane Lipids / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D021381:Protein Transport / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D008563:Membrane Lipids; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; D051057:Proto-Oncogene Proteins c-akt; D020764:cdc42 GTP-Binding Protein
keywords: Cdc42; PI3K; PIK3CA; microinjection; mutation; oncogene