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© The Rockefeller University Press, 0021-9525/1999//347 $5.00
The Journal of Cell Biology, Volume 145, Number 2, , 1999 347-361

Regular Articles

Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility



Yuko Fukata*, Noriko Oshiro*, Nagatoki Kinoshita*, Yoji Kawano*, Yoichiro Matsuoka{ddagger}, Vann Bennett{ddagger}, Yoshiharu Matsuura§, and Kozo Kaibuchi*

* Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan; {ddagger} Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710; and § Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan

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 migra- tion in NRK49F cells. {alpha}-AdducinT445D,T480D (substi- tution 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.

Key Words: Rho • Rho-kinase • adducin • membrane ruffling • cell motility

Abbreviations used in this paper: {alpha}-adducin-AA, {alpha}-adducin with substitution of Thr445 and Thr480 by Ala; {alpha}-adducin-DD, {alpha}-adducin with substitution of Thr445 and Thr480 by Asp; CAT, catalytic domain; ERM, ezrin/radixin/moesin; GST, glutathione-S-transferase; HA, hemagglutinin; HGF, hepatocyte growth factor; LPA, lysophosphatidic acid; MBP, maltose-binding protein; MBS, myosin-binding subunit; MLC, myosin light chain; PH, pleckstrin-homology domain; PK, protein kinase; RB, Rho-binding domain; TPA, tetradecanoylphorbol-13-acetate.


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