Sawada Takahiro, Jing Xuefeng, Zhang Yueqiang, Shimada Emi, Yokote Hideyuki, Miyajima Masayasu, Sakaguchi Kazushige
Department of Molecular Cell Biology and Molecular Medicine, Institute of Advanced Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, JapanLaboratory Animal Center, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan.
Genes Cells. 2010 Mar;15(3):297-311. doi: 10.1111/j.1365-2443.2010.01391.x. Epub 2010 Feb 24.
EphA4 belongs to a superfamily of receptor tyrosine kinases and interacts with several molecules including fibroblast growth factor receptors (FGFRs) as we reported earlier. Several receptor tyrosine kinases, FGFRs, Trks, Alk and Ret, are currently known to transduce a signal through a docking protein, fibroblast growth factor receptor substrate 2α (FRS2α). However, nothing has been reported about the interaction of FRS2α with EphA4. Using the yeast two-hybrid system and the in vitro binding and kinase assays, we found that the mid-kinase region of EphA4 directly interacts with the FRS2α PTB domain upon tyrosine phosphorylation of the EphA4 juxtamembrane (JM) domain and EphA4 directly phosphorylates FRS2α. We also found that the FRS2α PTB domain and the amino-terminal region of EphA4 bind to the amino- and carboxy-terminal regions of the FGFR JM domain, respectively, suggesting that FRS2α and EphA4 interact with FGFR simultaneously. Furthermore, a kinase-dead EphA4 mutant that constitutively binds to FGFR functions as a dominant-negative molecule for signaling through both EphA4 and FGFR, and so does the truncated FRS2α lacking multiple tyrosine phosphorylation sites. These dominant-negative mutants similarly inhibit the ligand-dependent proliferation of the mouse embryonic neural stem/progenitor cells. These results suggest the formation of a ternary complex comprising EphA4, FGFR and FRS2α. The signaling complex appears to integrate the input from FGFR and EphA4, and release the output signal through FRS2α.
正如我们之前报道的，EphA4属于受体酪氨酸激酶超家族，可与包括成纤维细胞生长因子受体（FGFRs）在内的多种分子相互作用。目前已知几种受体酪氨酸激酶，即FGFRs、Trks、Alk和Ret，可通过对接蛋白成纤维细胞生长因子受体底物2α（FRS2α）转导信号。然而，关于FRS2α与EphA4的相互作用尚无相关报道。利用酵母双杂交系统以及体外结合和激酶分析，我们发现，在EphA4近膜（JM）结构域发生酪氨酸磷酸化后，EphA4的中间激酶区域直接与FRS2α的PTB结构域相互作用，并且EphA4可直接使FRS2α磷酸化。我们还发现，FRS2α的PTB结构域和EphA4的氨基末端区域分别与FGFR JM结构域的氨基末端和羧基末端区域结合，这表明FRS2α和EphA4可同时与FGFR相互作用。此外，持续结合FGFR的激酶失活型EphA4突变体作为一种显性负性分子，可通过EphA4和FGFR发出信号，缺乏多个酪氨酸磷酸化位点的截短型FRS2α亦是如此。这些显性负性突变体同样可抑制小鼠胚胎神经干/祖细胞的配体依赖性增殖。这些结果表明，形成了一种由EphA4、FGFR和FRS2α组成的三元复合物。该信号复合物似乎整合了来自FGFR和EphA4的输入信号，并通过FRS2α释放输出信号。
