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Localized phosphorylation of vimentin by Rho‐kinase in neuroblastoma N2a cells
Author(s) -
Nakamura Yu,
Hashimoto Ryota,
Amano Mutsuki,
Nagata Kohichi,
Matsumoto Naohiko,
Goto Hidemasa,
Fukusho Eriko,
Mori Hiroshi,
Kashiwagi Yujiro,
Kudo Takashi,
Inagaki Masaki,
Takeda Masatoshi
Publication year - 2000
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.2000.00372.x
Subject(s) - neurite , vimentin , rho associated protein kinase , biology , microbiology and biotechnology , cytoskeleton , intermediate filament , phosphorylation , kinase , protein kinase a , neurofilament , in vitro , biochemistry , cell , immunology , immunohistochemistry
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 μ M Y‐27632, a Rho‐kinase specific inhibitor, abolished TM71 immuno‐reactivity, and induced irregular neurite outgrowth. In contrast, 20 n M 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 μ M 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.