Phosphorylation of ERM proteins at filopodia induced by Cdc42.
BACKGROUND
ERM (ezrin, radixin, and moesin) proteins function as membrane-cytoskeletal linkers, and are known to be localized at filopodia and microvilli-like structures. We have shown that Rho-associated kinase (Rho-kinase)/ROKalpha/ROCK II phosphorylates moesin at Thr-558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli-like structures (Oshiro, N., Fukata, Y. & Kaibuchi, K. (1998) Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273, 34663- 34666). However, the role of ERM proteins in the formation of filopodia is less well characterized.


RESULTS
Here we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)-terminal threonine. When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42V12), filopodia formation was induced and phosphorylation of ERM at C-terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells. We also showed that Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C-terminal threonine in a cell-free system. Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C-terminal threonine-phosphorylated ERM proteins at filopodia induced by Cdc42V12.


CONCLUSIONS
The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.
Nakamura
N
N
Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan.
Oshiro
N
N
Fukata
Y
Y
Amano
M
M
Fukata
M
M
Kuroda
S
S
Matsuura
Y
Y
Leung
T
T
Lim
L
L
Kaibuchi
K
K
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D005455:Fluorescent Antibody Technique; D005799:Genes, Dominant / Q000235:genetics / Q000502:physiology; D005982:Glutathione Transferase / Q000096:biosynthesis; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011554:Pseudopodia / Q000502:physiology*; D011994:Recombinant Proteins; D012097:Repressor Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000494:pharmacology*; D054460:rho-Associated Kinases; D000099149:Ezrin; D000099291:Adducins
D001798:Blood Proteins; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D005982:Glutathione Transferase; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013912:Threonine; D020764:cdc42 GTP-Binding Protein; D054460:rho-Associated Kinases; D000099149:Ezrin; D000099291:Adducins; C111391:HA 126-138; C111065:Mlc protein, E coli; C071058:moesin; C071776:radixin