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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 PI3K? (phosphoinositide 3-kinase ?) 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 PI3K? in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3K? is pre-formed as a 1:1 p110?:p85? 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? was recruited to the PM, but oncogenic forms of PI3K? were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3K?. 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.

SUBMITTER: Layton MJ

PROVIDER: S-EPMC3985441 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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

Layton Meredith J MJ Rynkiewicz Natalie K NK Ivetac Ivan I Horan Kristy A KA Mitchell Christina A CA Phillips Wayne A WA

Bioscience reports 20140401 2

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 PIP<sub>3</sub>/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 ...[more]

PMID: 27919038

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Project description:Nucleophosmin (NPM) is a nucleolar phosphoprotein that is involved in many cellular processes and has both oncogenic and growth suppressing activities. NPM is localized primarily in nucleoli but shuttles between the nucleus and the cytoplasm, and sustained cytoplasmic distribution contributes to its tumor promoting activities. Plakoglobin (PG, γ-catenin) is a homolog of β-catenin with dual adhesive and signaling functions. These proteins interact with cadherins and mediate adhesion, while their signaling activities are regulated by association with various intracellular partners. Despite these similarities, β-catenin has a well-defined oncogenic activity, whereas PG acts as a tumor/metastasis suppressor through unknown mechanisms. Comparison of the proteomic profiles of carcinoma cell lines with low- or no PG expression with their PG-expressing transfectants has identified NPM as being upregulated upon PG expression. Here, we examined NPM subcellular distribution and in vitro tumorigenesis/metastasis in the highly invasive and very low PG expressing MDA-MB-231 (MDA-231) breast cancer cells and their transfectants expressing increased PG (MDA-231-PG) or NPM shRNA (MDA-231-NPM-KD) or both (MDA-231-NPM-KD+PG). Increased PG expression increased the levels of nucleolar NPM and coimmunoprecipitation studies showed that NPM interacts with PG. PG expression or NPM knockdown decreased the growth rate of MDA-231 cells substantially and this reduction was decreased further in MDA-231-NPM-KD+PG cells. In in vitro tumorigenesis/metastasis assays, MDA-231-PG cells showed substantially lower and MDA-231-NPM-KD cells substantially higher invasiveness relative to the MDA-231 parental cells, and the co-expression of PG and NPM shRNA led to even further reduction of the invasiveness of MDA-231-PG cells. Furthermore, examination of the levels and localization of PG and NPM in primary biopsies of metastatic infiltrating ductal carcinomas revealed coordinated expression of PG and NPM. Together, the data suggest that PG may regulate NPM subcellular distribution, which may potentially change the function of the NPM protein from oncogenic to tumor suppression.

Dataset Information

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

Publications

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

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