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

作　　者：赵晓涛[1] 张正[1] 

机构地区：[1]北京大学人民医院检验科,100044

出　　处：《中华医学杂志》2006年第15期1052-1056,共5页National Medical Journal of China

摘　　要：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, a

关 键 词：神经肌肉接头 蛋白质酪氨酸磷酸酶 受体 胆碱能 

分 类 号：R33[医药卫生—人体生理学]