Article Figures & Data
Figures
- Figure 1 SHP2 inhibition by NSC-87877 induces MuSK phosphorylation and AChR clustering
(A) Dose-response curve of the effects of SHP2 inhibition on AChR phosphorylation. Phosphorylation increased progressively reaching a maximal effect at 100 µM and decreasing at higher concentrations. (B) Clustering of AChRs (labeled with Alexa Fluor 594 α-bungarotoxin) in C2C12 myotubes after 12 hours incubation with DMEM, agrin, or 100 μM NSC-87877. Both agrin and NSC-87877 increased AChR clusters in native C2C12 myotubes but not in MuSK knockout cells. The optimal NSC-87877 concentration was 100 µM, and cluster numbers decreased at higher concentrations. (C and D) The pooled results of 3–6 independent experiments measuring MuSK phosphorylation (C) and AChR clusters (D) are shown. (E) Time course of MuSK phosphorylation in C2C12 myotubes after exposure to agrin (1:800), NSC-87877 (100 μM), and agrin/NSC-87877. In each case, a similar time course was observed with maximum effect between 40 and 90 minutes. Phosphorylation and MuSK blots shown for NSC-87877 alone. All images are 20× magnification. Scale bar represents 50 µm. Mean + standard error of the mean are shown. Results were analyzed by 2-sided t tests. AChR = acetylcholine receptor; MuSK = muscle-specific kinase; SHP2 = SRC homology 2 domain-containing phosphotyrosine phosphatase 2.
- Figure 2 Effect of SHP2 inhibition in DOK7-overexpressing C2C12s
(A) MuSK phosphorylation was already high in DOK7-overexpressing C2C12s (data not shown) but was increased further by NSC-87877. (B) Equally DOK7-overexpressing myotubes showed AChR clusters that were further increased by NSC-87877. All images are 20× magnification. Scale bar represents 50 µm. Results were analyzed by 2-sided t tests. DOK7 = downstream of kinase 7; MuSK = muscle-specific kinase; SHP = SRC homology 2 domain-containing phosphotyrosine phosphatase 2.
- Figure 3 SHP2 inhibition reverses effects of MuSK-Abs on AChR clusters in C2C12s and DOK7-overexpressing C2C12s
(A) The treatment protocols are shown above the results. MuSK-Ab positive (n = 21) or healthy control sera (n = 2) were incubated with myotubes for 30 minutes, followed by agrin (1:800) with or without NSC-87877 (100 μM). After 12 hours, the AChR clusters were analyzed and expressed as a percentage of control serum results. The number (A.a) of the AChR clusters without NSC-87877 was clearly reduced by the MuSK-Abs (to 14%, 35%, and 60% at dilutions 1:10, 1:30, and 1:90, red columns); the size of the clusters was similarly reduced (A.b). In each case, the effects were partially or completely reversed by NSC-87877 (green columns). (B) Inverse correlation between the number of AChR clusters at 1:30 serum dilution and MuSK-Ab titers (nM) with or without NSC-87877. (C) Using a different protocol, AChR clusters were similarly reduced, and there was partial or complete protection by NSC-87877. (D) The effects of the different serum dilutions on AChR clusters and the protection by NSC-87877 were also seen in the DOK7-overexpressing C2C12s, although to a lesser extent. In A, C, and D, comparisons at each different serum concentration were analyzed by 2-sided t tests. For B, linear regression was computed by GraphPad Prism. The scatter plots show results of individual sera, with mean ± SDs. AChR = acetylcholine receptor; MuSK = muscle-specific kinase; SHP2 = SRC homology 2 domain-containing phosphotyrosine phosphatase 2.
- Figure 4 IgG and IgG4 subclass MuSK-Abs reduce AChR clusters, which are restored by NSC-87877
Agrin-stimulated myotubes were exposed for 40 minutes to MuSK IgG or IgG4 subfraction (adjusted to 0.5 nM MuSK-Ab) purified from 2 patients with MuSK-MG (the total number of experiments was IgG n = 5; IgG4 n = 6). (A.a) Example of phosphorylation blots. MuSK was immunoprecipitated by anti-MuSK AF562 or MuSK-MG IgG fractions. Pooled results (A.b) show that total IgG and IgG4 inhibited MuSK phosphorylation, and this was increased substantially by NSC-87877 (100 μM). (B.a) Examples of AChR clusters. Pooled results (B.b) show that total IgG and Ig4 MuSK-Ab fractions inhibited the formation of AChR clusters, which in each case were increased by NSC-87877. Two-sided t tests were used for comparisons at each IgG concentration. AChR = acetylcholine receptor; MG = Myasthenia gravis; MuSK = muscle-specific kinase.
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