Phosphorylation of Collapsin Response Mediator Protein-2 by Rho-kinase

We previously identified Rho-associated protein kinase(Rho- kinase) as a specific effector of Rho. In this study, we identified collapsin response mediator protein-2 (CRMP-2), as a novel Rho- kinasesubstrate in the brain. CRMP-2 is a neuronal protein whose expression is up-regulated during development. Rho- kinasephosphorylated CRMP-2 at Thr-555 in vitro. We produced an antibody that specifically recognizes CRMP-2 phosphorylated at Thr-555. Using this antibody, we found that Rho- kinasephosphorylated CRMP-2 downstream of Rho in COS7 cells. Phosphorylation of CRMP-2 was observed in chick dorsal root ganglionneurons during lysophosphatidic acid(LPA)-induced growth conecollapse, whereas the phosphorylation was not detected during semaphorin-3A-induced growth conecollapse. Both LPA-induced CRMP-2 phosphorylation and LPA-induced growth conecollapse were inhibited by Rho-kinase inhibitorHA1077 or Y-32885. LPA-induced growth conecollapse was also blocked by a dominant negative form of Rho- kinase. On the other hand, semaphorin-3Ainduced growth conecollapse was not inhibited by a dominant negative form of Rho- kinase. Furthermore, overexpression of a mutant CRMP-2 in which Thr-555 was replaced by Ala significantly inhibited LPA-induced growth conecollapse. These results demonstrate the existence of Rho- kinase-dependent and -independent pathways for growth conecollapse and suggest that CRMP-2 phosphorylation by Rho- kinaseis involved in the former pathway.