Agrin phosphorylates PI3-K, AKT, and GSK3β in muscle cells. (a) Neural, but not nonneural, agrin phosphorylates GSK3β via phosphorylation of PI3-K and AKT in cultured myotubes. Western blots of lysates from C2C12 myotubes to which soluble neural (5 nM) or nonneural (5 nM) agrin was added. Blots probed with antibodies as described in Materials and methods. Panels showing p-GSK3β and p-AKT are derived from same blot exposed for different times. Note that (1) AKT and GSK3β are not phosphorylated by nonneural agrin (top left panel, right lane); (2) their phosphorylation by neural agrin is inhibited by the PI3-K blocker LY294002 (50 µM), and the AKT inhibitor A6730 (0.5 µM, bottom left panel), respectively. Neural agrin also phosphorylates AKT and GSK3β when presented attached to culture substrate (top right panel, see Materials and methods). Full blots are shown in Fig. S1. Bar, 20 µm. (b) PI3-K is activated at the postsynaptic membrane of the NMJ. Soleus muscles of wild-type mice were electroporated with expression constructs for PH-BTK or PH-BTKmut, i.e., pleckstrin homology domain fragments of Brutton tyrosine kinase, which specifically bind (PH-BTK-GFP) or do not bind (PH-BTK_R28C-GFP) to PIP₃, a read-out for PI3-K activity. Both constructs were tagged with eGFP (see Várnai et al., 1999; Bohnacker et al., 2009). Top: longitudinal confocal sections passing through NMJs of muscle fibers expressing elevated GFP (green) at synaptic AChR cluster (BTX594, red). Bars, 10 µm. Bottom: cross sections through fibers electroporated with the two constructs. Synaptic localization of PH-BTK-GFP is higher than that of PH-BTKmut-GFP, indicating synaptic PI3-K activity. Graph shows means ± SEM of fold change in GFP intensity at synaptic AChR clusters at 10–14 d after electroporation with PH-BTK or PH-BTKmut, respectively (n = 20 synapses on GFP-positive fibers per construct examined, *, P < 0.05; **, P < 0.01, two-sided t test). Bars, 7.5 µm and 3 µm. (c) P-GSK3β is enriched at the NMJ. Immunoreactivity is abolished by pretreatment of muscle with lambda-phosphatase. Bar, 10 µm. For specificity of antibody used, see Fig. S2. (d) High resolution confocal image of p-GSK3β immunoreactivity. GSK3β-(Ser9)P labeling was not distributed evenly. Rather, p-GSK3β labeling was preferentially located between the crests of the synaptic folds carrying the AChRs, as is the case for CLIP-170 and CLASP2 (Fig. 2 a). Enlarged inset and corresponding line profiles of AChR and GSK3β-P fluorescence are shown on the right. Bars, 3 µm and 0.5 µm.