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

作者：Layton Meredith J; Rynkiewicz Natalie K; Ivetac Ivan; Horan Kristy A; Mitchell Christina A; Phillips Wayne A^*

来源：Bioscience Reports, 2014, 34(2): 165-+.

DOI：10.1042/BSR20130133

• Cdc42
• microinjection
• mutation
• oncogene
• PI3K
• PIK3CA

Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3K alpha (phosphoinositide 3-kinase alpha) 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 PI3Ka in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Ka is pre-formed as a 1:1 p110 alpha:p85 alpha 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 PI3K alpha was recruited to the PM, but oncogenic forms of PI3K alpha were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3K alpha. 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.

• 出版日期2014