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

Abstract

To investigate the involvement of protein tyrosine phosphatases Shp-2 in regulating postsynaptic signaling at the NMJ. Cultured C2 mouse myotubes were used to mimic NMJ formation; immunoprecipitation, immuno-blot, RNA interference and immunofluorescent labeling were used in this study. 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 cells: the MuSK band at 115 kD showed increased tyrosine phosphorylation after pervanadate treatment; 10 micromol/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. 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.