title: 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.
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas, USA.
authors.forename: Heather S
authors.initials: HS
authors.lastname: Carr
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Morris
authors.forename: Sarita
authors.initials: S
authors.lastname: Menon
authors.forename: Eun Hyeon
authors.initials: EH
authors.lastname: Song
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Frost
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D005260:Female; D022001:Focal Adhesions / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D051379:Mice; D018994:Myosin Light Chains / Q000378:metabolism; D015513:Oncogene Proteins / Q000032:analysis / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D054462:p21-Activated Kinases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D018994:Myosin Light Chains; C099763:NET1 protein, human; D015513:Oncogene Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; C039077:platelet protein P47; D054462:p21-Activated Kinases; D046988:Proteasome Endopeptidase Complex; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein