Phosphorylation of collapsin response mediator protein-2 by Rho-kinase - Evidence for two separate signaling pathways for growth cone collapse

We previously identified Rho-associated protein kinase (Rho-kinase) as a sp ecific effector of Rho, In this study, we identified collapsin response med iator protein-2 (CRMP-2), as a novel Rho-kinase substrate in the brain. CRM P-2 is a neuronal protein whose expression is up-regulated during developme nt. Rho-kinase phosphorylated CRMP-2 at Thr-555 in vitro. We produced an an tibody that specifically recognizes CRMP-2 phosphorylated at Thr-555, Using this antibody, we found that Rho-kinase phosphorylated CRMP-2 downstream o f Rho in COS7 cells. Phosphorylation of CRMP-2 was observed in chick dorsal root ganglion neurons during lysophosphatidic acid (LPA)-induced growth co ne collapse, whereas the phosphorylation was not detected during semaphorin -3A-induced growth cone collapse. Both LPA-induced CRMP-2 phosphorylation a nd LPA-induced growth cone collapse were inhibited by Rho-kinase inhibitor UA1077 or Y-32885. LPA-induced growth cone collapse was also blocked by a d ominant negative form of Rho-kinase. On the other hand, semaphorin-3A-induc ed growth cone collapse was not inhibited by a dominant negative form of Rh o-kinase, Furthermore, overexpression of a mutant CRMP-2 in which Thr-555 w as replaced by Ala significantly inhibited LPA-induced growth cone collapse . These results demonstrate the existence of Rho-kinase-dependent and -inde pendent pathways for growth cone collapse and suggest that CRMP-2 phosphory lation by Rho-kinase is involved in the former pathway.