Phosphorylation of collapsin response mediator protein-2 by Rho-kinase -: Evidence for two separate signaling pathways for growth cone collapse
Phosphorylation of collapsin response mediator protein-2 by Rho-kinase -: Evidence for two separate signaling pathways for growth cone collapse
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DOI：
10.1074/jbc.m001032200
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发表时间：
2000-08-04
影响因子：
4.8
通讯作者：
Kaibuchi, K
中科院分区：
文献类型：
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作者：
Arimura, N;Inagaki, N;Kaibuchi, K
我们先前发现Rho相关蛋白激酶(Rho-kinaseRho-kinase，Rho-Kinase)是Rho的特异效应物，在本研究中，我们发现了崩解素反应介体蛋白-2(CRMP-2)，它是大脑中一种新的Rho-Kinase底物。CRMP-2是一种神经元蛋白，在发育过程中表达上调。Rho-Kinase在体外使CRMP-2在Thr-555处磷酸化。我们制备了一种特异性识别Thr-555处磷酸化的CRMP-2的抗体，利用该抗体，我们发现在COS7细胞中，Rho-Kinase能够磷酸化Rho下游的CRMP-2。在溶血磷脂酸(LPA)诱导的生长锥塌陷过程中，鸡背根神经节神经元中观察到CRMP-2的磷酸化，而在信号素-3A诱导的生长锥塌陷过程中，未检测到CRMP-2的磷酸化。Rho-Kinase抑制剂UA1077或Y-32885均能抑制LPA诱导的CRMP-2磷酸化和LPA诱导的生长锥体塌陷。LPA诱导的生长锥塌陷也被显性的阴性形式的Rho-Kinase所阻断。另一方面，信号素-3A诱导的生长锥塌陷不能被显性的Rho-激酶负性形式所抑制，此外，Ala取代Thr-555的突变体CRMP-2的过表达也显著抑制了LPA诱导的生长锥塌陷。这些结果证明了生长锥塌陷存在依赖和非依赖的Rho-Kinase途径，并提示Rho-Kinase对CRMP-2的磷酸化参与了前者。
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 UA1077 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.