Localized phosphorylation of vimentin by rho-kinase in neuroblastoma N2a cells.
BACKGROUND
Vimentin, which is one of the intermediate filaments, is the major cytoskeletal component in developing neurones or neuroblastoma cells. Rho-associated kinase (Rho-kinase), is rich in neurones and is found downstream of Rho. It is involved in the agonist-induced neurite retraction of neuronal cells, and phosphorylates vimentin at Ser-38 and Ser-71 resulting in in vitro disassembly of the filaments.


RESULTS
We have investigated the distribution of vimentin phosphorylated by Rho-kinase in N2a neuroblastoma cells using site-specific phosphorylation-dependent antibodies. TM71 immunoreactivity, which specifically indicates Ser-71 phosphorylation on vimentin, was found in some neurites of dibutyryl cAMP-differentiated N2a cells. Transfection of the constitutively active form of Rho-kinase, CAT, significantly elevated TM71 immunoreactivity, and induced neurite retraction or cell rounding. Conversely, transfection of the dominant negative form of Rho-kinase, RB/PH(TT), or treatment of 10 microM Y-27632, a Rho-kinase specific inhibitor, abolished TM71 immuno-reactivity, and induced irregular neurite outgrowth. In contrast, 20 nM okadaic acid (OA) induced neurite retraction and specifically elevated TM71 immunoreactivity. In the OA-induced neurite retraction, tubulin disappeared in retracting neurites, where vimentin and actin remained co-localized. Furthermore, the OA-induced elevation of TM71 immunoreactivity and neurite retraction were completely blocked by pretreatment with 10 microM Y-27632, or by the ectopic expression of RB/PH(TT).


CONCLUSIONS
This study suggests that the localized phosphorylation of vimentin by Rho-kinase in neurites was closely related with the cellular morphology of N2a cells, and that the Rho-kinase activity towards vimentin was balanced with OA-sensitive phosphatases.
Nakamura
Y
Y
Department of Clinical Neuroscience, Psychiatry, Osaka University Graduate School of Medicine, Yamadaoka 565-0871, Japan. nakamura@psy.med.osaka-u.ac.jp
Hashimoto
R
R
Amano
M
M
Nagata
K
K
Matsumoto
N
N
Goto
H
H
Fukusho
E
E
Mori
H
H
Kashiwagi
Y
Y
Kudo
T
T
Inagaki
M
M
Takeda
M
M
D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D048429:Cell Size; D019084:Fluorescent Antibody Technique, Indirect; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D016501:Neurites / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000378:metabolism*; D054460:rho-Associated Kinases
D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D011725:Pyridines; D014746:Vimentin; C108830:Y 27632; D010768:Phosphoserine; D019319:Okadaic Acid; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases