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, Kozo Kaibuchi

Research output: Contribution to journalArticle

238 Citations (Scopus)

Abstract

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 α-adducin and thereby enhances the F-actin-binding activity of α-adducin in vitro. Here we identified the sites of phosphorylation of α-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-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 α-adducin(T445A,T480A) (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migration in NRK49F cells, α- Adducin(T445D,T480D) (substitution of Thr445 and Thr480 by Asp), but not α- 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 α-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.

Original languageEnglish
Pages (from-to)347-361
Number of pages15
JournalJournal of Cell Biology
Volume145
Issue number2
DOIs
Publication statusPublished - Jun 2000
Externally publishedYes

Keywords

  • Adducin
  • Cell motility
  • Membrane ruffling
  • Rho
  • Rho-kinase

ASJC Scopus subject areas

  • Cell Biology

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  • Cite this

    Fukata, Y., Oshiro, N., Kinoshita, N., Kawano, Y., Matsuoka, Y., Bennett, V., Matsuura, Y., & Kaibuchi, K. (2000). Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility. Journal of Cell Biology, 145(2), 347-361. https://doi.org/10.1083/jcb.145.2.347