Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility.
Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates alpha-adducin and thereby enhances the F-actin-binding activity of alpha-adducin in vitro. Here we identified the sites of phosphorylation of alpha-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized alpha-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated alpha-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated alpha-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or alpha-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migration in NRK49F cells. alpha-AdducinT445D,T480D (substitution of Thr445 and Thr480 by Asp), but not alpha-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates alpha-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
Fukata
Y
Y
Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan.
Oshiro
N
N
Kinoshita
N
N
Kawano
Y
Y
Matsuoka
Y
Y
Bennett
V
V
Matsuura
Y
Y
Kaibuchi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D002465:Cell Movement; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D004285:Dogs; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine; D054460:rho-Associated Kinases; D000099291:Adducins
D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D019204:GTP-Binding Proteins; D020690:GTPase-Activating Proteins; D017228:Hepatocyte Growth Factor; D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate; D013912:Threonine; D054460:rho-Associated Kinases; D000099291:Adducins; C085379:rho GTPase-activating protein