Abstract
Dok-7 is a cytoplasmic activator of muscle-specific receptor-tyrosine kinase (MuSK). Both Dok-7 and MuSK are required for neuromuscular synaptogenesis. Mutations in DOK7 underlie a congenital myasthenic syndrome (CMS) associated with small and simplified neuromuscular synapses likely due to impaired Dok-7/MuSK signaling. The overwhelming majority of patients with DOK7 CMS have at least one allele with a frameshift mutation that causes a truncation in the COOH-terminal region of Dok-7 and affects MuSK activation. Dok-7 has pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in the NH2-terminal moiety, both of which are indispensable for MuSK activation in myotubes, but little is known about additional functional elements. Here, we identify a chromosome region maintenance 1-dependent nuclear export signal (NES) in the COOH-terminal moiety and demonstrate that the NES-mediated cytoplasmic location of Dok-7 is essential for regulating the interaction with MuSK in myotubes. The NH2-terminal PH domain is responsible for the nuclear import of Dok-7. We also show that the Src homology 2 target motifs in the COOH-terminal moiety of Dok-7 are active and crucial for MuSK activation in myotubes. In addition, CMS-associated missense mutations found in the PH or PTB domain inactivate Dok-7. Together, these findings demonstrate that, in addition to the NH2-terminal PH and PTB domains, the COOH-terminal NES and Src homology 2 target motifs play key roles in Dok-7/MuSK signaling for neuromuscular synaptogenesis. Ablation or disruption of these functional elements in Dok-7 probably underlies the neuromuscular junction synaptopathy observed in DOK7 CMS.
Highlights
Dok-7 is the latest member of the Dok-family proteins, which share structural similarities characterized by the NH2-terminal
Antibodies, and Reagents—Constructions of human Dok-7, Dok-7- dupTGCC, Dok-7-RA (R158A, R159A, and R174A), Dok-7-dN [61–504], and mouse MuSK-myc expression plasmids were described elsewhere [1, 26]. cDNAs encoding Dok-7 mutants (Dok-7-R158Q, -A33V, -R201X, -N110, -1143insC, -N420, -Y395F, -Y405F, -2YF, -2PA, -L241A, -L245A, -L248A) were generated by PCR to be inserted into pcDNA3.1-myc/His (Invitrogen) or pEGFP-N3 (Clontech), which is an expression plasmid for an Myc/His-tagged protein or enhanced green fluorescent protein (EGFP) fusion, respectively. cDNAs encoding NES and mutant NES of Dok-7 were generated by PCR and inserted into pEGFP-C1 (Clontech)
Two missense mutations, 98C3 T and 473G3 A, which produce mutant Dok-7 harboring A33V and R158Q substitutions in the PH and PTB domains, respectively, have been found in patients with DOK7 CMS (Fig. 1A and Refs. 27 and 28). Both Ala-33 and Arg-158 are conserved among vertebrates, and Dok-7-A33V and Dok-7-R158Q failed to induce MuSK activation, as judged by tyrosine phosphorylation of MuSK and its downstream target acetylcholine receptors (AChRs)1 and subsequent AChR clustering in myotubes (Fig. 1, B–E)
Summary
Antibodies, and Reagents—Constructions of human Dok-7, Dok-7- dupTGCC, Dok-7-RA (R158A, R159A, and R174A), Dok-7-dN [61–504], and mouse MuSK-myc expression plasmids were described elsewhere [1, 26]. cDNAs encoding Dok-7 mutants (Dok-7-R158Q, -A33V, -R201X, -N110, -1143insC, -N420, -Y395F, -Y405F, -2YF, -2PA, -L241A, -L245A, -L248A) were generated by PCR to be inserted into pcDNA3.1-myc/His (Invitrogen) or pEGFP-N3 (Clontech), which is an expression plasmid for an Myc/His-tagged protein or enhanced green fluorescent protein (EGFP) fusion, respectively. cDNAs encoding NES and mutant NES (mNES) of Dok-7 were generated by PCR and inserted into pEGFP-C1 (Clontech). CDNAs encoding Dok-7 mutants (Dok-7-R158Q, -A33V, -R201X, -N110, -1143insC, -N420, -Y395F, -Y405F, -2YF, -2PA, -L241A, -L245A, -L248A) were generated by PCR to be inserted into pcDNA3.1-myc/His (Invitrogen) or pEGFP-N3 (Clontech), which is an expression plasmid for an Myc/His-tagged protein or enhanced green fluorescent protein (EGFP) fusion, respectively. CDNAs encoding NES and mutant NES (mNES) of Dok-7 were generated by PCR and inserted into pEGFP-C1 (Clontech). The following antibodies were from commercial sources: anti-␣-tubulin (DM1A), anti-Dok-7 (H-77), anti-MuSK (N-19 and C-19), antiphosphorylated AChR1 (Tyr-390), anti-green fluorescent protein (B-2), anti-myosin heavy chain (F59), and horseradish peroxidase (HRP)-conjugated anti-goat IgG (Santa Cruz Biotechnology); anti-MuSK (AF562) (R&D Systems); anti-phosphotyrosine (4G10) (Upstate Biotechnology); HRP-conjugated anti-rabbit, rat, or mouse IgG (GE Healthcare); anti-Myc (9B11) (Cell Signaling Technology); anti-CrkII monoclonal antibody (BD Transduction Laboratories).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have