Phosphorylation of actopaxin regulates cell spreading and migration.
Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.
Clarke
Dominic M
DM
Department of Cell Biology and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.
Brown
Michael C
MC
LaLonde
David P
DP
Turner
Christopher E
CE
D000185:Actinin; D000595:Amino Acid Sequence; D002070:Butadienes / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D048429:Cell Size / Q000235:genetics; D002999:Clone Cells; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D000096985:Calponins
D000185:Actinin; D002070:Butadienes; D004791:Enzyme Inhibitors; D008840:Microfilament Proteins; D009570:Nitriles; C486590:PARVA protein, human; D000096985:Calponins; C113580:U 0126