J. Cell Biol., Volume 145, Number 2, April 19, 1999 347-361

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

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

^* Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan;  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 -adducin and thereby enhances the F-actin-binding activity of -adducin in vitro. Here we identified the sites of phosphorylation of -adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized -adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated -adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated -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 -adducin^T445A,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. -Adducin^T445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not -adducin^T445A,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 -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.

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