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Mutations Causing DOK7 Congenital Myasthenia Ablate Functional Motifs in Dok-7

2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês

10.1074/jbc.m708607200

1083-351X

Johko Hamuro, Osamu Higuchi, Kumiko Okada, Makiko Ueno, Shun‐ichiro Iemura, Tohru Natsume, Hayley Spearman, David Beeson, Yuji Yamanashi,

Ion channel regulation and function

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. 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. Dok-7 is the latest member of the Dok-family proteins, which share structural similarities characterized by the NH2-terminal PH 3The abbreviations used are:PHpleckstrin homologyPTBphosphotyrosine bindingBtxα-bungarotoxinLMBleptomycin BIPimmunoprecipitationIBimmunoblottingMuSKmuscle-specific receptor-tyrosine kinaseCMScongenital myasthenic syndromeSH2Src homology 2NMJneuromuscular junctionAChRacetylcholine receptorNESnuclear export signalEGFPenhanced green fluorescent proteinpYphosphotyrosineWCLwhole cell lysates. and PTB domains followed by the SH2 target motifs in the COOH-terminal moiety, suggesting an adaptor function (1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar, 2Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 3Carpino N. Wisniewski D. 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CMS comprise a heterogeneous group of NMJ disorders associated with genetic defects in presynaptic, synaptic, and in most cases, postsynaptic proteins of the NMJ (10Engel A.G. Ohno K. Sine S.M. Nat. Rev. Neurosci. 2003; 4: 339-352Crossref PubMed Scopus (154) Google Scholar, 11Beeson D. Hantaï D. Lochmüller H. Engel A.G. Neuromuscul. Disord. 2005; 15: 498-512Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Engel A.G. Neurotherapeutics. 2007; 4: 252-257Crossref PubMed Scopus (97) Google Scholar). Impaired synaptic transmission at the NMJ results in fatigable muscle weakness and typically may variably affect limb, ocular, bulbar, truncal, and respiratory muscles. 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Neuromuscul. Disord. 2005; 15: 498-512Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24Slater C.R. Fawcett P.R. Walls T.J. Lyons P.R. Bailey S.J. Beeson D. Young C. Gardner-Medwin D. Brain. 2006; 129: 2061-2076Crossref PubMed Scopus (97) Google Scholar, 25Engel A.G. Brain. 2006; 129: 1938-1939Crossref PubMed Scopus (6) Google Scholar). Recently, we found biallelic mutations in DOK7 underlie a major subgroup of CMS with predominantly proximal muscle weakness that did not show tubular aggregates on muscle biopsy but were found to have normal AChR function but abnormally small and simplified NMJs (26Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. Müller J.S. Lochmüller H. Slater C. Vincent A. Yamanashi Y. Science. 2006; 313: 1975-1978Crossref PubMed Scopus (233) Google Scholar). Patients with DOK7 mutations do not usually show long-term benefit from anticholinesterase medication (26Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. Müller J.S. Lochmüller H. Slater C. Vincent A. Yamanashi Y. Science. 2006; 313: 1975-1978Crossref PubMed Scopus (233) Google Scholar, 27Palace J. Lashley D. Newsom-Davis J. Cossins J. Maxwell S. Kennett R. Jayawant S. Yamanashi Y. Beeson D. Brain. 2007; 130: 1507-1515Crossref PubMed Scopus (102) Google Scholar, 28Müller J.S. Herczegfalvi A. Vilchez J.J. Colomer J. Bachinski L.L. Mihaylova V. Santos M. Schara U. Deschauer M. Shevell M. Poulin C. Dias A. Soudo A. Hietala M. Aärimaa T. Krahe R. Karcagi V. Huebner A. Beeson D. Abicht A. Lochmüller H. Brain. 2007; 130: 1497-1506Crossref PubMed Scopus (128) Google Scholar), but ephedrine can be helpful (24Slater C.R. Fawcett P.R. Walls T.J. Lyons P.R. Bailey S.J. Beeson D. Young C. Gardner-Medwin D. Brain. 2006; 129: 2061-2076Crossref PubMed Scopus (97) Google Scholar, 27Palace J. Lashley D. Newsom-Davis J. Cossins J. Maxwell S. Kennett R. Jayawant S. Yamanashi Y. Beeson D. Brain. 2007; 130: 1507-1515Crossref PubMed Scopus (102) Google Scholar). The relatively late onset of presenting symptoms in some of these patients may indicate that Dok-7 plays a role not only in the formation but also in the maintenance of NMJ structure. Also of note, all cases of DOK7 CMS reported to date have been found to harbor at least one allele with a mutation in the COOH-terminal coding exon of DOK7, suggesting a pathophysiological importance for this region which has yet to be analyzed in detail for functional elements (26Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. Müller J.S. Lochmüller H. Slater C. Vincent A. Yamanashi Y. Science. 2006; 313: 1975-1978Crossref PubMed Scopus (233) Google Scholar, 27Palace J. Lashley D. Newsom-Davis J. Cossins J. Maxwell S. Kennett R. Jayawant S. Yamanashi Y. Beeson D. Brain. 2007; 130: 1507-1515Crossref PubMed Scopus (102) Google Scholar, 28Müller J.S. Herczegfalvi A. Vilchez J.J. Colomer J. Bachinski L.L. Mihaylova V. Santos M. Schara U. Deschauer M. Shevell M. Poulin C. Dias A. Soudo A. Hietala M. Aärimaa T. Krahe R. Karcagi V. Huebner A. Beeson D. Abicht A. Lochmüller H. Brain. 2007; 130: 1497-1506Crossref PubMed Scopus (128) Google Scholar). Here, we use the AChR clustering pathway in C2 myotubes, where the Dok-7/MuSK signaling plays an essential role as in vivo, in association with DOK7 CMS mutations (1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar, 26Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. Müller J.S. Lochmüller H. Slater C. Vincent A. Yamanashi Y. Science. 2006; 313: 1975-1978Crossref PubMed Scopus (233) Google Scholar, 27Palace J. Lashley D. Newsom-Davis J. Cossins J. Maxwell S. Kennett R. Jayawant S. Yamanashi Y. Beeson D. Brain. 2007; 130: 1507-1515Crossref PubMed Scopus (102) Google Scholar, 28Müller J.S. Herczegfalvi A. Vilchez J.J. Colomer J. Bachinski L.L. Mihaylova V. Santos M. Schara U. Deschauer M. Shevell M. Poulin C. Dias A. Soudo A. Hietala M. Aärimaa T. Krahe R. Karcagi V. Huebner A. Beeson D. Abicht A. Lochmüller H. Brain. 2007; 130: 1497-1506Crossref PubMed Scopus (128) Google Scholar), to reveal important functional modules in Dok-7. We show that a NES and two SH2 target motifs in the COOH-terminal region are essential for Dok-7 to activate MuSK. In accordance, CMS-associated mutant Dok-7 lacking both elements fails to activate MuSK. Given that the PH domain, which is indispensable for MuSK activation, has a nuclear localization activity, the NES-mediated cytoplasmic retention of Dok-7 likely plays a role in regulating interaction with MuSK at the juxtamembrane part of cells. In addition, we show that CMS-associated missense mutations found in the PH or PTB domain inactivate Dok-7. Together, these findings suggest multiple pathogenic mechanisms underlying DOK7 CMS. Plasmids, 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 (1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar, 26Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. Müller J.S. Lochmüller H. Slater C. Vincent A. Yamanashi Y. Science. 2006; 313: 1975-1978Crossref PubMed Scopus (233) Google Scholar). 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). Mouse CrkII expression plasmids, pcDNA3-mCrkII and pcDNA3-mCrkII-R38K, were kindly provided from Dr. Hidesaburo Hanafusa (Osaka Bioscience Institute). The following antibodies were from commercial sources: anti-α-tubulin (DM1A), anti-Dok-7 (H-77), anti-MuSK (N-19 and C-19), anti-phosphorylated AChRβ1 (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). Anti-Dok-7 rat anti-sera raised against peptide for the COOH-terminal portion of human Dok-7 (214-498) were prepared as described elsewhere (1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar). Leptomycin B was from Biomol. Alexa 594-conjugated α-bungarotoxin (Btx) and 4,6-diamidino-2-phenylindole were from Molecular Probes. Immunoprecipitation, Immunoblotting, Btx Pulldown, Fluorescent Microscopy, MuSK Phosphorylation, and AChR Clustering Assays—C2 myoblasts (C2C12) were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum. After transfection with Dok-7 expression plasmids using FuGENE 6 (Roche Applied Science), C2 myoblasts were cultured in differentiation medium (Dulbecco's modified Eagle's medium supplemented with 2% horse serum) for 5 days. We previously demonstrated that exogenous Dok-7 proteins were expressed in both undifferentiated myoblasts and differentiated myotubes (1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar). Differentiated myotubes were subjected to immunoprecipitation, immunoblotting, and Btx pulldown assays as described (1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar). Note that Btx-Sepharose was used to pull down the AChR pentamer complex, which includes the AChRβ1 subunit. For AChR clustering assays, 10 microscopic fields with the 40× objective were chosen at random, and the numbers of AChR clusters (>5 μm in their longest diameter) visualized with Alexa 594-conjugated Btx were counted using a DM6000B microscope (Leica). For fluorescent microscopy assays in Figs. 2 and 3, digital images were analyzed with Leica Deblur® software (AutoQuant) in a FW4000Z imaging system (Leica). For the MuSK phosphorylation assay in Fig. 4D, phosphorylated MuSK in C2 myotubes was visualized by immunoprecipitation and immunoblotting as in Fig. 4B, and levels of each phosphorylation were quantified with densitometry using NIH Image software (Version 1.63).FIGURE 3The NES-like sequence of Dok-7 serves as a functional NES and is crucial for MuSK activation in myotubes. A, the core Leu-241 in the NES-like sequence is required for the nuclear export of Dok-7-EGFP (WT). L241A, but not L248A substitution inhibited the nuclear export of Dok-7-EGFP. L245A substitution showed partial effects. Scale bar, 20 μm. B, the NES-like sequence facilitates accumulation of EGFP in the cytoplasm. EGFP fused with the 27 amino acids (225-251) of human Dok-7 including the NES-like sequence (NES-EGFP), but not its mutant form (mNES-EGFP) harboring a substitution equivalent to L241A mutation, was excluded from the nucleus (arrowheads). NES-EGFP translocated into the nucleus after the LMB treatment. Scale bar, 20 μm. C and D, the NES of Dok-7 is crucial for MuSK activation and AChR clustering. Indicated proteins were exogenously expressed in C2 myotubes, and cells were subjected to fluorescent microscopy (A and B) as in Fig. 2 or to IP/IB (C) or the AChR-clustering assay (D) as in Fig. 1. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4The SH2 target motifs in Dok-7 COOH-terminal moiety are crucial for MuSK activation in myotubes. A, two SH2 target motifs (boxed) in the COOH-terminal portion of Dok-7 are conserved among human, mouse, and fish. Pink-shading indicates amino acids with 100% identity; red and blue asterisks indicate the core tyrosine and proline residues, respectively (upper panel). CMS-associated mutants Dok-7-dupTGCC (dupTGCC) and Dok-7-1143insC (1143insC), but not an unrelated mutant Dok-7-N420, lack the SH2 target motifs (lower panel). YY and green boxes indicate the SH2 target motifs and the polypeptides generated by the frameshift mutations, respectively. WT, wild type. h, m, f, human, mouse, and fish, respectively. B-E, the core residues of the SH2 target motifs are crucial for MuSK activation and AChR clustering in myotubes. Dok-7 proteins indicated were exogenously expressed in C2 myotubes, and cells were subjected to IP/IB (B) or a AChR clustering assay (C and E) as in Fig. 1 or to a MuSK phosphorylation assay (D). Relative intensity of MuSK phosphorylation (mean ± S.D.; n = 3) is shown (D), where the intensity of MuSK phosphorylation in C2 myotubes transfected with empty plasmid was defined as 1.0 in the arbitrary units. Tub, tubulin. F and G, the polypeptide (421-504) COOH-terminal to the SH2 target motifs is dispensable for Dok-7 to activate MuSK in myotubes. Dok-7-N420, but not Dok-7-1143insC, normally induced MuSK activation (F) and AChR clustering (G). Dok-7 proteins indicated were exogenously expressed in C2 myotubes, and cells were subjected to IP/IB (F) or AChR clustering assay (G) as in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A33V or R158Q Missense Mutations in the PH or PTB Domains Inhibit Dok-7-mediated Activation of MuSK—Previously we demonstrated that the PH and PTB domains of Dok-7 are essential for MuSK activation in cultured myotubes (1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar). However, it has remained unclear whether these domains play crucial roles in MuSK-dependent postsynaptic differentiation in vivo. Recently, two missense mutations, 98C→ T and 473G→ 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. 27Palace J. Lashley D. Newsom-Davis J. Cossins J. Maxwell S. Kennett R. Jayawant S. Yamanashi Y. Beeson D. Brain. 2007; 130: 1507-1515Crossref PubMed Scopus (102) Google Scholar and 28Müller J.S. Herczegfalvi A. Vilchez J.J. Colomer J. Bachinski L.L. Mihaylova V. Santos M. Schara U. Deschauer M. Shevell M. Poulin C. Dias A. Soudo A. Hietala M. Aärimaa T. Krahe R. Karcagi V. Huebner A. Beeson D. Abicht A. Lochmüller H. Brain. 2007; 130: 1497-1506Crossref PubMed Scopus (128) Google Scholar). 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 AChRβ1 and subsequent AChR clustering in myotubes (Fig. 1, B-E). We confirmed that the forced expression of Dok-7 does not affect the differentiation of myotubes with regard to morphology and expression of myosin heavy chain, a known differentiation marker (supplemental Fig. S1 and Ref. 29Cabane C. Englaro W. Yeow K. Ragno M. Dérijard B. Am. J. Physiol. Cell Physiol. 2003; 284: 658-666Crossref PubMed Scopus (82) Google Scholar). Thus, these CMS-associated mutations inactivate Dok-7, which apparently is mediated through an effect on the PH or PTB domain. Indeed, Dok-7-R158Q showed the same defect, a failure to bind with MuSK in HEK 293T cells, as Dok-7-RA, which carries an inactivated PTB domain (Fig. 1F and Ref. 1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar). It should be noted that the PTB domain is necessary for Dok-7 to bind with MuSK (but not to activate MuSK) in heterologous cells, whereas it is required to activate MuSK in myotubes (Fig. 1, C, E, and F, and Ref. 1Okada K. Inoue A. Okada M. Murata Y. Kakuta S. Jigami T. Kubo S. Shiraishi H. Eguchi K. Motomura M. Akiyama T. Iwakura Y. Higuchi O. Yamanashi Y. Science. 2006; 312: 1802-1805Crossref PubMed Scopus (337) Google Scholar). Therefore, we conclude that the 473G→ A mutation found in a patient with DOK7 CMS disrupts the PTB domain. Together, these data suggest crucial roles of the PH and PTB domains of Dok-7 in humans. Dok-7 Shuttles between the Cytoplasm and the Nucleus in Myotubes—601C→ T is a nonsense mutation identified in a patient with DOK7 CMS that produces a mutant peptide (Dok-7-R201X) lacking the entire COOH-terminal region (Arg-201 to Pro-504) (Fig. 2A and Refs. 26Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. Müller J.S. Lochmüller H. Slater C. Vincent A. Yamanashi Y. Science. 2006; 313: 1975-1978Crossref PubMed Scopus (233) Google Scholar and 27Palace J. Lashley D. Newsom-Davis J. Cossins J. Maxwell S. Kennett R. Jayawant S. Yamanashi Y. Beeson D. Brain. 2007; 130: 1507-1515Crossref PubMed Scopus (102) Google Scholar). Interestingly, Dok-7-R201X completely failed to activate MuSK and induce subsequent AChR clustering in myotubes (Fig. 2, B and C), unlike mutant Dok-7 produced by frameshift mutation 1124_1127dupTGCC (Dok-7-dupTGCC) which lacks the COOH-terminal peptide from Ala-378 to Pro-504 but induces reduced, but significant levels of MuSK activation as well as AChR clustering (Fig. 2A and Ref. 26Beeson D. Higuchi O. Palace J. Cossins J. Spearman H. Maxwell S. Newsom-Davis J. Burke G. Fawcett P. Motomura M. Müller J.S. Lochmüller H. Slater C. Vincent A. Yamanashi Y. Science. 2006; 313: 1975-1978Crossref PubMed Scopus (233) Google Scholar). Thus, there is possibly an additional functional element in the COOH-terminal region between Arg-201 and Ala-377. By searching databases, we noticed a potential NES sequence composed of 10 amino acids (240-249 amino acids) including Leu-241, Leu-245, and Leu-248 in human Dok-7, which are conserved among vertebrates (Fig. 2, A and D). We, therefore, investigated subcellular localization of Dok-7-R201X in myotubes to determine whether Dok-7 shuttles between the cytoplasm and the nucleus. Dok-7-R201X showed prominent accumulation in the nucleus of cultured myotubes, whereas the wild-type control was mainly in the cytoplasm (Fig. 2E), suggesting that the NES-like sequence is functional. Indeed, leptomycin B (LMB), a specific inhibitor of the CRM1-dependent nuclear exporter, induced nuclear accumulation of Dok-7 (Fig. 2F). Although we failed to find a canonical nuclear localization signal sequence in Dok-7, a mutant harboring a large NH2-terminal deletion in the PH domain (dN-EGFP) was cytoplasmic even in the presence of LMB (Fig. 2F). Conversely, the PH domain (1-110 amino acids) of Dok-7 fused with EGFP (N110-EGFP) strongly accumulated in the nucleus, indicating that the PH domain of Dok-7 has a nuclear localization activity. Taken together, these findings suggest that Dok-7 shuttles between