Activation of Rho family small G proteins is thought to be a critical event in breast cancer development and metastatic progression. Rho protein activation is stimulated by a family of enzymes known as guanine nucleotide exchange factors (Rho GEFs). The neuroepithelioma transforming gene 1 (Net1) is a Rho GEF specific for the RhoA subfamily that is overexpressed in primary breast tumors and breast cancer cell lines. Net1 isoform expression is also required for migration and invasion of breast cancer cells in vitro. These data indicate that Net1 may be a critical regulator of metastatic progression in breast cancer. Net1 activity is negatively regulated by sequestration in the nucleus, and relocalization of Net1 outside the nucleus is required to stimulate RhoA activation, actin cytoskeletal reorganization, and oncogenic transformation. However, regulatory mechanisms controlling the extranuclear localization of Net1 have not been identified. In this study, we have addressed the regulation of Net1A isoform localization by Rac1. Specifically, co-expression of constitutively active Rac1 with Net1A stimulates the relocalization of Net1A from the nucleus to the plasma membrane in breast cancer cells, and results in Net1A activation. Importantly, Net1A localization is also driven by endogenous Rac1 activity. Net1A relocalizes outside the nucleus in cells spreading on collagen, and when endogenous Rac1 expression was silenced by siRNA, Net1A remained nuclear in spreading cells. These data indicate that Rac1 controls the localization of the Net1A isoform and suggests a physiological role for Net1A in breast cancer cell adhesion and motility
