Chen Ting-Huan, Chen Chen-Yu, Wen Hui-Chin, Chang Chia-Chu, Wang Horng-Dar, Chuu Chih-Pin, Chang Chung-Ho
Department of Life Science, National Tsing Hua University, Hsin-Chu, Taiwan, China.
Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan, China.
FASEB J. 2017 Jul;31(7):2963-2972. doi: 10.1096/fj.201601090R. Epub 2017 Mar 29.
Yes-associated protein (YAP) is a transcriptional coactivator in the Hippo pathway that regulates cell proliferation, differentiation, and apoptosis. The MEK5/ERK5 MAPK cascade is essential for the early step of myogenesis. In this study, we generated C2C12 stable cell lines that expressed YAP (C2C12-YAP cells) and found that ERK5 and MEK5 were activated in C2C12-YAP cells compared with control C2C12 (C2C12-vector) cells. C2C12-YAP stable cells also differentiated into myotubes better than C2C12-vector cells, and expressed elevated levels of myogenin, a transcription factor that regulates myogenesis, as well as elevated levels of myosin heavy chain, a skeletal muscle marker. Western blot analysis revealed that Src and c-Abl (Abelson murine leukemia viral oncogene homolog 1) activation were enhanced in C2C12-YAP cells. Conversely, treatment of inhibitors of c-Abl, Src, or MEK5 inhibited activation of MEK5 and ERK5 and myogenesis of C2C12 myoblasts. Specific interactions between YAP and proteins in the ERK5 pathway, such as MEK kinase 3 (MEKK3) and ERK5, were illustrated by coimmunoprecipitation experiments. MEKK3 contains the PPGY motif (aa 178-181), which may interact with YAP. Site-directed mutagenesis experiments revealed that expression of MEKK3 Y181F mutant inhibited MEK5/ERK5 activation and myogenic differentiation. These results suggest that YAP promotes muscle differentiation by activating the Abl/Src/MEKK3/MEK5/ERK5 kinase cascade.-Chen, T.-H., Chen, C.-Y., Wen, H.-C., Chang, C.-C., Wang, H.-D., Chuu, C.-P., Chang, C.-H. YAP promotes myogenic differentiation the MEK5-ERK5 pathway.
Yes相关蛋白（YAP）是Hippo信号通路中的一种转录共激活因子，可调节细胞增殖、分化和凋亡。MEK5/ERK5丝裂原活化蛋白激酶级联反应对于成肌作用的早期步骤至关重要。在本研究中，我们构建了表达YAP的C2C12稳定细胞系（C2C12-YAP细胞），并发现与对照C2C12（C2C12-载体）细胞相比，C2C12-YAP细胞中的ERK5和MEK5被激活。C2C12-YAP稳定细胞分化为肌管的能力也比C2C12-载体细胞更强，并且肌生成素（一种调节成肌作用的转录因子）以及骨骼肌标志物肌球蛋白重链的表达水平升高。蛋白质印迹分析显示，C2C12-YAP细胞中Src和c-Abl（阿贝尔逊鼠白血病病毒癌基因同源物1）的激活增强。相反，用c-Abl、Src或MEK5的抑制剂处理可抑制MEK5和ERK5的激活以及C2C12成肌细胞的成肌作用。共免疫沉淀实验表明YAP与ERK5信号通路中的蛋白（如MEK激酶3（MEKK3）和ERK5）之间存在特异性相互作用。MEKK3含有PPGY基序（第178-181位氨基酸），可能与YAP相互作用。定点诱变实验表明，MEKK3 Y181F突变体的表达抑制了MEK5/ERK5的激活和成肌分化。这些结果表明，YAP通过激活Abl/Src/MEKK3/MEK5/ERK5激酶级联反应促进肌肉分化。-陈，T.-H.，陈，C.-Y.，温，H.-C.，张，C.-C.，王，H.-D.，朱，C.-P.，张，C.-H. YAP通过MEK5-ERK5信号通路促进成肌分化
