Rac1 Controls the Subcellular Localization of the Rho Guanine Nucleotide Exchange Factor Net1A To Regulate Focal Adhesion Formation and Cell Spreading

Abstract

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.

Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity

“…Notably, mutation of both NLS sequences together caused a significant relocalization of Net1A outside the nucleus that was as strong as that caused by coexpression of constitutively active V 12 Rac1, which potently stimulates Net1A localization to the cytosol and plasma membrane (Fig. 1E,F) (Carr et al, 2013a). Taken together, these data indicate that Net1 isoforms contain additional, unrecognized NLS sequences that contribute significantly to their nuclear localization.…”

“…Recently we have shown that activation of Rac1 downstream of integrin ligation causes a robust, temporally regulated relocalization of the Net1A isoform to the plasma membrane. Moreover, Net1A relocalization is required for focal adhesion maturation and cell spreading in breast cancer cells (Carr et al, 2013a). However, the mechanism by which Rac1 stimulates Net1A relocalization is not known.…”

“…Net1 isoforms localize within the nucleus in quiescent cells, thereby preventing them from activating RhoA (Carr et al, 2013a;Qin et al, 2005;Schmidt and Hall, 2002). Previously it has been shown that the longer Net1 isoform contains two NLS sequences within its unique N-terminal regulatory domain.…”

“…It has been shown previously that breast cancer cells mainly require Net1A, but not Net1, for adhesion, motility and ECM invasion (Carr et al, 2013a;Carr et al, 2013b;Dutertre et al, 2010). Moreover, Rac1 preferentially stimulates relocalization of Net1A, but not Net1, to the plasma membrane (Carr et al, 2013a).…”

“…Net1 proteins are unusual among Rho GEFs in that they localize to the nucleus in resting cells (Carr et al, 2013a;Qin et al, 2005;Schmidt and Hall, 2002). However, it is clear that Net1 isoforms must be exported to the plasma membrane to stimulate RhoA activity and initiate actin cytoskeletal reorganization Schmidt and Hall, 2002).…”