Roles of ShcB/ShcC Docking Proteins in Neural Stem Cells and Neuronal Tumors

Shc是Shc对接蛋白家族的一员，它含有独特的PTB-CH1-SH2模块化结构，并作为各种受体酪氨酸激酶的底物进行传导。最近，我们发现在一些神经母细胞系，如NB-39-nu细胞中检测到的过度磷酸化的ShcC与由该基因扩增引起的结构性激活的间变性淋巴瘤激酶(ALK)有关。在约10%的原发人类神经母细胞瘤中也检测到ALK基因扩增。SiRNA抑制Nb-39-nu细胞中ALK的表达，导致ShcC的磷酸化水平降低，MAPKIAkt途径失活，细胞凋亡，提示ALK酪氨酸激酶是该神经母细胞瘤细胞系的主要生存信号。为了研究过度磷酸化的ShcC在神经母细胞瘤细胞系中的作用，我们建立了过表达野生型和突变型ShcC蛋白的NB-39-nu细胞系。结果表明，表达ShcC 3YF突变体的NB-39-nu细胞的细胞存活、分化和运动能力明显受损，该突变体以显性-负性方式阻断了ShcC-Grb2通路。同时，MAPK和Akt的激活水平在这些细胞中受到严重抑制。另一方面，过度表达ShcC的细胞和3YF突变体表现出转化能力降低，如锚定独立性和体内致瘤性，这可能表明ShcC特异性的负面作用。在悬浮细胞培养中，在ShcC高表达细胞和3YF细胞中都观察到pl3OCas持续磷酸化的丧失。这些结果表明，除了MAPK和Akt途径外，ShcC可能还对另一种途径进行负调控，如Src家族激酶(SFK)-p130Cas途径。

ShoC is a family member of Shc docking proteins, which contain a unique PTB-CH1-SH2 modular organization and conduct as substrates of various receptor tyrosine kinases. Recently, we showed that hyperphosphorylated ShcC detected in some of neuroblastorna cell lines, such as NB-39-nu cells, is associated with constitutively activated anaplastic lymphoma kinase (ALK) caused by the gene amplification. The ALK gene amplification was also detected in about 10% of primary human neuroblastomas. Suppression of ALK expression in NB-39-nu cells by siRNA resulted in decreased phosphorylation level of ShcC, inactivation of MAPKIAkt pathway and cell apoptosis suggesting that ALK tyrosine kinase is dominating survival signal of this neuroblastoma line. To investigate the roles of hyperphosphorylated ShcC in neuroblastoma cell lines, we established NB-39-nu cell lines which overexpress wildtype or mutant ShcC proteins. It was demonstrated that cell-survival and differentiation, cell-motility were markedly impaired in the NB-39-nu cells expressing the 3YF mutant of ShcC which blocks ShcC-Grb2 pathway by the dominant-negative fassion. At the same time, activation level of MAPK and Akt was severely suppressed in these cells. On the other hand, cells overexpressing ShcC as well as the 3YF mutant showed decreased transforming ability, such as anchorage independency and in vivo tumorigenicity that might suggest ShcC-specific negative effects. Loss of persistent phosphorylation of pl3OCas in suspension cell culture was observed in both ShcC overexpressing cells and 3YF cells. These results suggest ShcC might negatively regulate an alternative pathway such as the Src family kinase (SFK)-p130cas pathway in addition to the authentic MAPK and Akt pathways.