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

J Biol Chem. 2000 Aug 4;275(31):23973-80. doi: 10.1074/jbc.M001032200.

Abstract

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-kinase substrate in the brain. CRMP-2 is a neuronal protein whose expression is up-regulated during development. Rho-kinase phosphorylated 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-kinase phosphorylated CRMP-2 downstream of Rho in COS7 cells. Phosphorylation of CRMP-2 was observed in chick dorsal root ganglion neurons during lysophosphatidic acid (LPA)-induced growth cone collapse, whereas the phosphorylation was not detected during semaphorin-3A-induced growth cone collapse. Both LPA-induced CRMP-2 phosphorylation and LPA-induced growth cone collapse were inhibited by Rho-kinase inhibitor HA1077 or Y-32885. LPA-induced growth cone collapse was also blocked by a dominant negative form of Rho-kinase. On the other hand, semaphorin-3A-induced growth cone collapse 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 cone collapse. These results demonstrate the existence of Rho-kinase-dependent and -independent pathways for growth cone collapse and suggest that CRMP-2 phosphorylation by Rho-kinase is involved in the former pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies
  • Brain Chemistry
  • COS Cells
  • Cattle
  • Ganglia, Spinal / cytology
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Intracellular Signaling Peptides and Proteins
  • Lysophospholipids / pharmacology
  • Molecular Sequence Data
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / immunology
  • Nerve Tissue Proteins / metabolism*
  • Nerve Tissue Proteins / pharmacology
  • Neurons / drug effects
  • Neurons / physiology*
  • Phosphoproteins / immunology
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Proteins / metabolism
  • Semaphorin-3A
  • Substrate Specificity
  • rho-Associated Kinases

Substances

  • Antibodies
  • Intercellular Signaling Peptides and Proteins
  • Intracellular Signaling Peptides and Proteins
  • Lysophospholipids
  • Nerve Tissue Proteins
  • Phosphoproteins
  • Recombinant Proteins
  • Semaphorin-3A
  • collapsin response mediator protein-2
  • Protein-Serine-Threonine Kinases
  • rho-Associated Kinases