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 mu M 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 mu 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.