Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L

Abstract

Agrin, released by motor neurons, promotes neuromuscular synapse formation by stimulating MuSK, a receptor tyrosine kinase expressed in skeletal muscle. Phosphorylated MuSK recruits docking protein-7 (Dok-7), an adaptor protein that is expressed selectively in muscle. In the absence of Dok-7, neuromuscular synapses fail to form, and mutations that impair Dok-7 are a major cause of congenital myasthenia in humans. How Dok-7 stimulates synaptic differentiation is poorly understood. Once recruited to MuSK, Dok-7 directly stimulates MuSK kinase activity. This unusual activity of an adapter protein is mediated by the N-terminal region of Dok-7, whereas most mutations that cause congenital myasthenia truncate the C-terminal domain. Here, we demonstrate that Dok-7 also functions downstream from MuSK, and we identify the proteins that are recruited to the C-terminal domain of Dok-7. We show that Agrin stimulates phosphorylation of two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins: Crk and Crk-L. Furthermore, we show that selective inactivation of Crk and Crk-L in skeletal muscle leads to severe defects in neuromuscular synapses in vivo, revealing a critical role for Crk and Crk-L downstream from Dok-7 in presynaptic and postsynaptic differentiation.

Figure 1.

Agrin stimulates phosphorylation of Dok-7 Y396 and Y406. (A) Dok-7 was immunoprecipitated from lysates of C2 myotubes, which had been stimulated with Agrin for 30 min, or from 293 cells transfected with Dok-7 and MuSK. Following SDS-PAGE, Dok-7 was digested with trypsin, and the tryptic fragments were analyzed by LTQ-Orbitrap MS. Agrin stimulated phosphorylation of Y406 in C2 myotubes; both Y396 and Y406 were phosphorylated in 293 cells transfected with Dok-7 and MuSK. (B) Agrin stimulated phosphorylation of Y396 and Y406 in C2 myotubes. (C) Western blots of lysates (WCL) from transfected 293 cells show that Dok-7 was not detectably phosphorylated at Y348 or Y355, indicating that Y396 and Y406 are the only tyrosine phosphorylation sites in reconstituted 293 cells (n = 3; mean ± SEM). (D) Y396 and Y406 are the major tyrosine phosphorylation sites in Dok-7 expressed in C2 myotubes. Mutation of either Y396 or Y406 reduced the extent of Agrin-independent Dok-7 phosphorylation. Mutation of both residues reduced phosphorylation to background levels, indicating that Y396 and Y406s are the major, if not only, tyrosine phosphorylation sites. See also Supplemental Figure S1.

Figure 2.

Dok-7 tyrosine phosphorylation is dispensable for MuSK and AChR-β subunit tyrosine phosphorylation. (A) Dok-7 was overexpressed ∼100-fold in each C2 muscle cell line (n = 3; mean ± SEM). (B) MuSK phosphorylation levels were elevated ∼18-fold in each of the Dok-7 tyrosine mutant lines, compared with mock-infected C2 cells. MuSK phosphorylation levels were modestly higher (∼30%) in the cell line infected with wild-type Dok-7 than in cell lines expressing any of the Dok-7 tyrosine mutations (n = 3; mean ± SEM), indicating that Dok-7 stimulates MuSK activation largely through a mechanism that is independent of Dok-7 phosphorylation. (C) Like MuSK phosphorylation, AChR phosphorylation levels were induced to the same extent in cell lines expressing single or double mutations in Dok-7 and at levels that were modestly reduced (∼30%) compared with cells infected with wild-type Dok-7 (n = 3; mean ± SEM).

Figure 3.

Dok-7 tyrosine phosphorylation plays a critical role in AChR clustering. (A,B) Overexpression of wild-type Dok-7 stimulated AChR clustering (∼50-fold), independently of Agrin. Mutation of either Y396 or Y406 impaired AChR clustering, and mutation of both residues reduced AChR clustering still further. Mutation of Y396 or Y406 also led to a reduction in the size of AChR clusters and the density of AChRs within the cluster. Wild-type and mutant proteins were expressed at similar levels (Fig. 2A).

Figure 4.

Dok-7 Y396 and Y406 phosphopeptides bind to Crk-L and Crk in myotube lysates. (A) The amino acid sequences of the biotinylated Dok-7 phosphopeptides. (B) The Dok-7 biotin-pY406 phosphopeptide purified an ∼37-kDa doublet. The doublet failed to bind the nonphosphorylated biotin-Y406 peptide, and binding to the biotin-pY406 phosphopeptide was inhibited by addition of excess phosphopeptide. The ∼37-kDa doublet was identified as Crk-II and Crk-L by Q-TOF MS. (C) Western blots of myotube proteins isolated by binding to pY396 or pY406 phosphopeptides were probed with antibodies to Nck1/2, Crk-I, Crk-II, Crk-L, Abl, or Arg. Both phosphopeptides bound Crk-I, Crk-II, and Crk-L; in addition, the pY406 phosphopeptide bound Nck1/2. Neither phosphopeptide bound Abl or Arg. See also Supplemental Figure S2.

Figure 5.

Agrin stimulates recruitment of Crk-L and Crk to a MuSK/Dok-7 signaling complex. (A) Agrin stimulation of C2 myotubes led to the formation of a signaling complex between phosphorylated Dok-7 and Crk-L. Furthermore, two tyrosine phosphorylated proteins, Dok-7, and a 70-kDa protein (*), coimmunoprecipitated with Crk-L. (B) NCK1/2 and Dok7 do not coimmunoprecipitate. (C) Agrin stimulated the formation of a complex containing Dok-7, Crk-I, and Crk-II. See also Supplemental Figure S3.

Figure 6.

Crk-L and Crk are enriched at neuromuscular synapses. Cross-sections of innervated and denervated adult rat gastrocnemius muscles were stained with Alexa-594 α-BGT, which labels synapses (red), and antibodies to Crk-L or Crk-I/II (green). Crk-L (A) and Crk-I/II (B) staining were enriched at synaptic sites. (C) Crk-L was concentrated in the postsynaptic membrane, since staining remained at denervated synaptic sites.

Figure 7.

Crk and Crk-L play critical roles in neuromuscular synapse formation. (A,B) Muscles from E18.5 mice deficient in muscle Crk/Crk-L contained fewer synapses than wild-type mice, and these synapses were broadly distributed in the muscle (Supplemental Fig. S5). (C) Indeed, approximately one-half of myofibers in Crk/Crk-L-deficient muscle lacked innervation, as AChR clusters were absent over the entire myofiber length. For A and B, n = 3, and error bars indicate standard errors of the means. For C, n = 2. See also Supplemental Figure S5.

Figure 8.

Neuromuscular synapses are aberrant in Crk- and Crk-L-deficient muscle. In Crk/Crk-L-deficient muscle, synapses were unusually small (arrows in A), and the density of synaptic AChRs was reduced (B). (C) In Crk/Crk-L-deficient muscle, motor axons contacted AChR clusters but failed to stop growing. For A and B, n = 3, and error bars indicate standard errors of the means. For C, n = 2.