Uchida Yutaka, Ohshima Toshio, Yamashita Naoya, Ogawara Miyuki, Sasaki Yukio, Nakamura Fumio, Goshima Yoshio
Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
J Biol Chem. 2009 Oct 2;284(40):27393-401. doi: 10.1074/jbc.M109.000240. Epub 2009 Aug 3.
Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.
塌陷反应介导蛋白2（CRMP2）是一种细胞内蛋白，可介导轴突导向分子3A（Sema3A）的信号传导，该分子具有排斥性。Fyn是一种Src型酪氨酸激酶，参与Sema3A信号传导。然而，在该信号通路中CRMP2与Fyn之间的关系仍不清楚。在我们的研究中，我们证明了Fyn在HEK293T细胞中使CRMP2的酪氨酸（Tyr）32位点发生磷酸化。使用针对CRMP酪氨酸（Tyr）32位点的磷酸化特异性抗体进行免疫组织化学分析表明，酪氨酸（Tyr）32磷酸化的CRMP在包括背根神经节神经元、成年小脑分子层和浦肯野细胞层以及海马伞在内的神经系统中含量丰富。在背根神经节神经元中过表达CRMP2的非磷酸化突变体（酪氨酸（Tyr）32突变为苯丙氨酸（Phe）32）会干扰Sema3A诱导的生长锥塌陷反应。这些结果表明，Fyn依赖的CRMP2酪氨酸（Tyr）32位点磷酸化参与了Sema3A信号传导。
