Arimura N, Inagaki N, Chihara K, Ménager C, Nakamura N, Amano M, Iwamatsu A, Goshima Y, Kaibuchi K
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.
J Biol Chem. 2000 Aug 4;275(31):23973-80. doi: 10.1074/jbc.M001032200.
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.
我们之前鉴定出Rho相关蛋白激酶（Rho激酶）是Rho的一种特异性效应器。在本研究中，我们鉴定出塌陷反应介导蛋白2（CRMP-2）是脑中一种新的Rho激酶底物。CRMP-2是一种神经元蛋白，其表达在发育过程中上调。Rho激酶在体外使CRMP-2的苏氨酸555位点磷酸化。我们制备了一种特异性识别苏氨酸555位点磷酸化的CRMP-2的抗体。利用该抗体，我们发现在COS7细胞中Rho激酶在Rho下游使CRMP-2磷酸化。在溶血磷脂酸（LPA）诱导的鸡背根神经节神经元生长锥塌陷过程中观察到CRMP-2的磷酸化，而在semaphorin-3A诱导的生长锥塌陷过程中未检测到该磷酸化。Rho激酶抑制剂HA1077或Y-32885可抑制LPA诱导的CRMP-2磷酸化以及LPA诱导的生长锥塌陷。Rho激酶的显性负性形式也可阻断LPA诱导的生长锥塌陷。另一方面，Rho激酶的显性负性形式不能抑制semaphorin-3A诱导的生长锥塌陷。此外，苏氨酸555被丙氨酸取代的突变型CRMP-2的过表达显著抑制LPA诱导的生长锥塌陷。这些结果证明了生长锥塌陷存在Rho激酶依赖性和非依赖性途径，并提示Rho激酶介导的CRMP-2磷酸化参与了前一种途径。
