ABSTRACT: 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.
