Publications

Abstract

Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wildtype (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signaling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR overexpressing and estrogen receptor (ER) -negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca2+- and EGF- inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6 binding domain inhibits Ras/MAPK activity. AnxA6 knock-down in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca2+- and EGF-inducible manner and Fluorescence Resonance Energy Transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR overexpressing and ER-negative breast cancer cells.