RhoA is overexpressed in human
cancer and contributes to aberrant cell motility and metastatic progression; however, regulatory mechanisms controlling RhoA activity in
cancer are poorly understood. Neuroepithelial transforming gene 1 (Net1) is a RhoA
guanine nucleotide exchange factor that is overexpressed in human
cancer. It encodes two
isoforms, Net1 and Net1A, which cycle between the nucleus and plasma membrane. Net1
proteins must leave the nucleus to activate RhoA, but mechanisms controlling the extranuclear localization of Net1
isoforms have not been described. Here, we show that Rac1 activation causes relocalization of Net1
isoforms outside the nucleus and stimulates Net1A catalytic activity. These effects do not require Net1A catalytic activity, its pleckstrin homology domain, or its regulatory C terminus. We also show that Rac1 activation protects Net1A from
proteasome-mediated degradation. Replating cells on
collagen stimulates endogenous Rac1 to relocalize Net1A, and inhibition of
proteasome activity extends the duration and magnitude of Net1A relocalization. Importantly, we demonstrate that Net1A, but not Net1, is required for cell spreading on
collagen,
myosin light chain phosphorylation, and focal adhesion maturation. These data identify the first physiological mechanism controlling the extranuclear localization of Net1
isoforms. They also demonstrate a previously unrecognized role for Net1A in regulating cell adhesion.