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PMID: 10209029 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility.

The Journal of cell biology ·Vol. 145 ·No. 2 ·1999-04-19 ·Pages 347-61

Fukata Y, Oshiro N, Kinoshita N, Kawano Y, Matsuoka Y, Bennett V, Matsuura Y, Kaibuchi K

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 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.

MeSH Terms
Amino Acid Sequence Animals Calmodulin-Binding Proteins/chemistry,metabolism Cell Line Cell Membrane/drug effects,physiology,ultrastructure Cell Movement Cytoskeletal Proteins/metabolism Cytoskeleton/drug effects,physiology,ultrastructure Dogs GTP-Binding Proteins/metabolism GTPase-Activating Proteins Hepatocyte Growth Factor/pharmacology Humans Intracellular Signaling Peptides and Proteins Kidney Models, Biological Molecular Sequence Data Oligopeptides/chemical synthesis,chemistry,metabolism Peptide Fragments/chemistry,isolation & purification Phosphorylation Protein Serine-Threonine Kinases/metabolism Recombinant Proteins/chemistry,metabolism Tetradecanoylphorbol Acetate/pharmacology Threonine rho-Associated Kinases
Chemicals
Calmodulin-Binding Proteins Cytoskeletal Proteins GTPase-Activating Proteins Intracellular Signaling Peptides and Proteins Oligopeptides Peptide Fragments Recombinant Proteins adducin rho GTPase-activating protein Threonine Hepatocyte Growth Factor Protein Serine-Threonine Kinases rho-Associated Kinases GTP-Binding Proteins Tetradecanoylphorbol Acetate
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Fukata Y
Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan.
Oshiro N
Kinoshita N
Kawano Y
Matsuoka Y
Bennett V
Matsuura Y
Kaibuchi K
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.145.2.347
Published
1999-04-19
Pages
347-61
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2133101
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.145.2.347 PMC Full Text View journal details
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