Lai Kwok-On, Chen Yu, Po Hoi-Man, Lok Ka-Chun, Gong Ke, Ip Nancy Y
Department of Biochemistry, Molecular Neuroscience Center and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, People's Republic of China.
J Biol Chem. 2004 Apr 2;279(14):13383-92. doi: 10.1074/jbc.M313356200. Epub 2004 Jan 15.
Eph receptors and their cognate ligands ephrins are important players in axon guidance and neural patterning during development of the nervous system. Much of our knowledge about the signal transduction pathways triggered by Eph receptors has been related to the modulation of actin cytoskeleton, which is fundamental in mediating the cellular responses in growth cone navigation, cell adhesion, and cell migration. In contrast, little was known about whether long term activation of Eph receptor would regulate gene expression. Here we report a novel signaling pathway of EphA4, which involves activation of the tyrosine kinase Jak2 and the transcriptional activator Stat3. Transfection of COS7 cells with EphA4, but not the kinase-dead mutant, induced tyrosine phosphorylation of Jak2, Stat1, and Stat3. Treatment of cultured C2C12 myotubes with ephrin-A1 also induced tyrosine phosphorylation of Stat3, which was abolished by the Jak2 inhibitor AG490. Moreover, Jak2 was co-immunoprecipitated with EphA4 in muscle, and both proteins were concentrated at the neuromuscular junction (NMJ) of adult muscle. By using microarray analysis, we have identified acetylcholinesterase, the critical enzyme that hydrolyzed the neurotransmitter acetylcholine at the NMJ, as a downstream target gene of the Jak/Stat pathway in muscle. More importantly, ephrin-A1 increased the expression of acetylcholinesterase protein in C2C12 myotubes, which was abolished by AG490. In contrast, ephrin-A1 reduced the expression of fibronectin mRNA in C2C12 myotubes independently of Jak2. Finally, the expression level of acetylcholinesterase in limb muscle of EphA4 null mice was significantly reduced compared with the wild-type control. Taken together, these results have identified Jak/Stat proteins as the novel downstream targets of EphA4 signaling. In addition, the present study provides the first demonstration of a potential function of Eph receptors and Jak/Stat proteins at the NMJ.
Eph受体及其同源配体ephrin是神经系统发育过程中轴突导向和神经模式形成的重要参与者。我们对Eph受体触发的信号转导途径的许多了解都与肌动蛋白细胞骨架的调节有关，肌动蛋白细胞骨架在介导生长锥导航、细胞粘附和细胞迁移中的细胞反应方面至关重要。相比之下，关于Eph受体的长期激活是否会调节基因表达却知之甚少。在此我们报告了EphA4的一条新的信号通路，该通路涉及酪氨酸激酶Jak2和转录激活因子Stat3的激活。用EphA4而非激酶失活突变体转染COS7细胞，可诱导Jak2、Stat1和Stat3的酪氨酸磷酸化。用ephrin-A1处理培养的C2C12肌管也可诱导Stat3的酪氨酸磷酸化，而Jak2抑制剂AG490可消除这种磷酸化。此外，Jak2在肌肉中与EphA4共免疫沉淀，且这两种蛋白都集中在成年肌肉的神经肌肉接头（NMJ）处。通过微阵列分析，我们已确定乙酰胆碱酯酶是在NMJ处水解神经递质乙酰胆碱的关键酶，它是肌肉中Jak/Stat通路的下游靶基因。更重要的是，ephrin-A1增加了C2C12肌管中乙酰胆碱酯酶蛋白的表达，而AG490可消除这种增加。相比之下，ephrin-A1独立于Jak2降低了C2C12肌管中纤连蛋白mRNA的表达。最后，与野生型对照相比，EphA4基因敲除小鼠肢体肌肉中乙酰胆碱酯酶的表达水平显著降低。综上所述，这些结果已确定Jak/Stat蛋白是EphA4信号的新下游靶标。此外，本研究首次证明了Eph受体和Jak/Stat蛋白在NMJ处的潜在功能。
