含Shp-2的酪氨酸蛋白磷酸酶在神经肌肉接头形成中的作用

The role of protein tyrosine phosphatases involved in Shp-2 in the formation of the neuromuscular junction

目的研究酪氨酸蛋白磷酸酶及含SH2结构域的酪氨酸蛋白磷酸酶(Shp-2)在神经肌肉接头形成过程中的信号调控作用。方法培养小鼠C2C12成肌细胞并使之分化为成熟的肌管细胞,应用免疫沉淀、免疫印记、RNA阻断(RNAi)以及显微荧光等技术检测酪氨酸磷酸酶、Shp-2在肌特异性受体酪氨酸激酶(MuSK)活化、乙酰胆碱受体(AChR)成簇过程中的作用。结果应用酪氨酸蛋白磷酸酶的抑制剂过钒酸盐(pervanadate)使MuSK活性增加,同时增加了非聚集蛋白(agrin)依赖的AChR聚集,与对照组(0·96簇/细胞)比较增加了6·7倍(6·43簇/细胞)(P<0·01);同时过钒酸盐也增加agrin依赖的AChR聚集;通过免疫印记筛选显示Shp-2是小鼠肌肉细胞的主要酪氨酸蛋白磷酸酶;阻断Shp-2蛋白表达后,MuSK活性以及AChR聚集增加。结论包括Shp-2在内的酪氨酸蛋白磷酸酶信号被抑制时,MuSK活性以及AChR聚集增加;被激活时则MuSK活性以及AChR聚集降低。

Objective To investigate the involvement of protein tyrosine phosphatases Shp-2 in regulating postsynaptic signaling at the NMJ. Methods Cultured C2 mouse myotubes were used to mimic NMJ formation; immunoprecipitation, immuno-blot, RNA interference and immunofluorescent labeling were used in this study. Results We first showed that the general tyrosine phosphatase inhibitor pervanadate functionally activated MuSK and enhanced both agrin-independent and agrin-dependent AChR clustering in muscle ceils： the MuSK band at 115 kD showed increased tyrosine phosphorylation after pervanadate treatment; 10 μmol/L pervanadate increased AChR clustering （mean = 6. 43/cell） more than six-fold compared with control （ mean = 0. 96/cell ）, P 〈 0. 0001, t-test; inclusion of pervanadate with agrin increased the size of agrin-induced clusters（difference = 1.53-fold, P 〈0. 0001, t-test） without significantly increasing the number of clusters （P = 0. 08, t-test）. Next, by immuno-screening we identified the SH2 domain-containing phosphatase Shp2 as a major tyrosine phosphatase in C2 myotubes and demonstrated that its selective down-regulation by RNA interference increased MuSK activation and AChR clustering： MuSK phosphorylation observed in the presence of pervanadate alone was increased in Shp2-depleted cells relative to control cells; AChR clusters in untreated myotubes were counted and data pooled from four experiments showed a doubling of their number （ 2. 21-fold ） in cells transfected with the Shp2 siRNA （ n = 198 ） compared to those transfected with the control siRNA （n = 220） ; the number of AChR clusters was also increased in Shp2 siRNA-transfected cells compared to controls following treatment with pervanadate （ 1.5- fold; n=149 cells） and agrin （1.41-fold; n = 125）, P〈0.001, t-test. We also asked how Shp2 affect AChR clustering after increasing Shp2 activity： C2C12 cells were transfected with Shp-2 active form （E76A） and wild type Shp2 （used as control） respectively, average length of AChR clusters in active form （E76A） was decreased about 20% compared with wild type Shp2, P 〈 0. 01, t-test. Conclusion These results suggest that Shp2 functions in a feedback loop to regulate agrin/MuSK signaling and these findings highlight the importance of protein tyrosine phosphatases in postsynaptic signaling at the NMJ and provide insights into how balancing actions of protein kinases and phosphatases may determine the formation of synaptic specializations in the developing nervous system.