YAP调控Dvl影响Wnt通路及肺癌恶性表型的分子机制

It is still controversial that YAP（Yes-associated protein）functions as a tumor suppressor or oncoprotein. Our previous study demonstrated that YAP located in cytoplasm could inhibit phosphorylation and nuclear import of Dvl, whereas YAP located in nuclei could upregulate phosphorylation and nuclear import of Dvl. This result indicated that YAP might influence Wnt signaling pathway by regulating phosphorylation and nuclear import of Dvl. However, the mechanism still remains unclear. Given the nuclear localization sequence (NLS) of Dvl was located within the PDZ domain and PY motif, we think that phosphorylated YAP located in cytoplasm could bind to the PDZ domain and PY motif of Dvl, and may block the NLS of Dvl, then influence the nuclear import of Dvl. Furthermore, the binding of YAP with the PDZ domain of Dvl may inhibit the binding of PDZ domain of Dvl with CK1δ/ε, which could inhibit the phosphorylation of Dvl. In contrast, non-phosphorylated YAP located in nuclei could not bind to Dvl, the binding of Dvl with CK1ε was significantly increased, and then the level of phosphorylation of Dvl could be increased. Also, the blocking of NLS of Dvl was depleted, and then the nuclear import of Dvl was increased. To test the hypothesis, we will construct and transfect YAP phosphorylation plasmid and YAP phosphorylated site mutant plasmid (make YAP located in cytoplasm/nuclei), YAP, CK1ε and Dvl plasmids or truncated mutants, and detect the molecular mechanism of YAP influencing Wnt signaling pathway by regulating phosphorylation and nuclear import of Dvl by adopting immunofluorescence, co-Immunoprecipitation, GST-pull down and western blot. These results may provide experimental basis for the prevention and treatment of lung cancer proliferation, invasion and metastasis.

YAP（Yes-associated protein）发挥促癌还是抑癌作用存有争议。我们发现YAP定位浆/核内时能分别抑制及促进Dvl磷酸化和入核水平，猜想YAP可能通过影响Dvl磷酸化及定位对Wnt通路发挥双重作用，但具体机制并不清楚。因Dvl的入核序列位于其PDZ结构域与PY基序之间，我们设想磷酸化YAP定位于浆内与PDZ和PY结合封闭了其入核序列，并占据了Dvl与激酶CK1ε生物结合位点PDZ，从而抑制了Dvl的磷酸化及入核。而去磷酸化YAP入核则解除了与Dvl的结合及入核序列抑制,因此增加了Dvl磷酸化及入核。本研究拟构建并转染YAP磷酸化及突变质粒（使其定位于浆/核）、YAP、CK1ε与Dvl结合的剪切体等，通过免疫荧光、IP、GST-pull down及免疫印迹等方法，证实YAP调控Dvl磷酸化及入核作用而影响Wnt通路的分子机制，为防治肺癌的增殖、侵袭提供实验基础。

Hippo信号通路为近年来识别的能够调节器官大小与细胞增殖的一条重要信号通路。LATS为丝氨酸/酪氨酸激酶,并被认为是Hippo通路的重要成员。YAP蛋白则为Hippo通路的最重要的下游效应因子。YAP核定位表达在包括肺癌在内的许多肿瘤中被发现。作为上游激酶，LATS能够导致YAP的磷酸化及浆内定位。另外，在之前的研究我们已经发现Wnt信号通路在非小细胞肺癌存在不同程度的激活，Dvl蛋白为Wnt信号通路的一个重要蛋白。并且我们发现Dvl蛋白的各个亚型，尤其是 Dvl2 在非小细胞肺癌存在明显上调。在本研究中，我们通过构建YAP与Dvl2相互作用的剪切体，然后通过转染、干扰、免疫共沉淀、免疫荧光及荧光素酶活性检测等方法探讨了LATS1和YAP对Dvl2蛋白的作用，并进一步探讨了Wnt及Hippo信号通路可能的相互作用。.结果显示，YAP经LATS1磷酸化后能够抑制Wnt信号通路的活性。磷酸化之后的YAP定位于胞浆，影响Dvl2蛋白的磷酸化及Dvl2蛋白的入核。进一步研究发现，YAP与CK1ε能够竞争性结合Dvl2的PDZ结构域，从而发挥抑制Wnt信号的作用。该研究能够使我们更好的理解Wnt通路与Hippo在肿瘤中的作用及两者的交互作用。