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 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.
Arimura
N
N
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.
Inagaki
N
N
Chihara
K
K
Ménager
C
C
Nakamura
N
N
Amano
M
M
Iwamatsu
A
A
Goshima
Y
Y
Kaibuchi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001923:Brain Chemistry; D019556:COS Cells; D002417:Cattle; D005727:Ganglia, Spinal / Q000166:cytology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000494:pharmacology; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D040121:Semaphorin-3A; D013379:Substrate Specificity; D054460:rho-Associated Kinases
D000906:Antibodies; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D040121:Semaphorin-3A; C095114:collapsin response mediator protein-2; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases