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

ShoC是Shc对接蛋白家族的成员之一，具有独特的PTB-CH 1-SH 2模块结构，是多种受体酪氨酸激酶的底物。最近，我们发现在一些成神经细胞系，如NB-39-nu细胞中检测到的过度磷酸化ShcC与由基因扩增引起的组成性激活的间变性淋巴瘤激酶（ALK）有关。在约10%的原发性人神经母细胞瘤中也检测到ALK基因扩增。通过siRNA抑制NB-39-nu细胞中的ALK表达导致ShcC磷酸化水平降低、MAPKIAkt通路失活和细胞凋亡，表明ALK酪氨酸激酶是该神经母细胞瘤系的主导存活信号。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，我们建立了过度表达野生型或突变型ShcC蛋白的NB-39-nu细胞系。结果表明，在表达ShcC的3 YF突变体的NB-39-nu细胞中，细胞存活和分化以及细胞运动明显受损，该突变体通过显性负性fassion阻断ShcC-Grb 2通路。同时，MAPK和Akt的活化水平在这些细胞中被严重抑制。另一方面，过表达ShcC以及3 YF突变体的细胞显示出降低的转化能力，如锚定独立性和体内致瘤性，这可能表明ShcC特异性的负面影响。在ShcC过表达细胞和3 YF细胞中观察到悬浮细胞培养物中pl 3 OCas的持续磷酸化的丧失。这些结果表明，ShcC可能负调节替代途径，如Src家族激酶（SFK）-p130 cas途径除了真实的MAPK和Akt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.