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• W2007826893 abstract "Myotubularin is the archetype of a family of highly conserved protein-tyrosine phosphatase-like enzymes. The myotubularin gene, MTM1, is mutated in the genetic disorder, X-linked myotubular myopathy. We and others have previously shown that myotubularin utilizes the lipid second messenger, phosphatidylinositol 3-phosphate (PI(3)P), as a physiologic substrate. We demonstrate here that the myotubularin-related protein MTMR2, which is mutated in the neurodegenerative disorder, type 4B Charcot-Marie-Tooth disease, is also highly specific for PI(3)P as a substrate. Furthermore, the MTM-related phosphatases MTMR1, MTMR3, and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this substrate is common to all myotubularin family enzymes. A direct comparison of the lipid phosphatase activities of recombinant myotubularin and MTMR2 demonstrates that their enzymatic properties are indistinguishable, indicating that the lack of functional redundancy between these proteins is likely to be due to factors other than the utilization of different physiologic substrates. To this end, we have analyzed myotubularin and MTMR2 transcripts during induced differentiation of cultured murine C2C12 myoblasts and find that their expression is divergently regulated. In addition, myotubularin and MTMR2 enhanced green fluorescent protein fusion proteins exhibit overlapping but distinct patterns of subcellular localization. Finally, we provide evidence that myotubularin, but not MTMR2, can modulate the levels of endosomal PI(3)P. From these data, we conclude that the developmental expression and subcellular localization of myotubularin and MTMR2 are differentially regulated, resulting in their utilization of specific cellular pools of PI(3)P. Myotubularin is the archetype of a family of highly conserved protein-tyrosine phosphatase-like enzymes. The myotubularin gene, MTM1, is mutated in the genetic disorder, X-linked myotubular myopathy. We and others have previously shown that myotubularin utilizes the lipid second messenger, phosphatidylinositol 3-phosphate (PI(3)P), as a physiologic substrate. We demonstrate here that the myotubularin-related protein MTMR2, which is mutated in the neurodegenerative disorder, type 4B Charcot-Marie-Tooth disease, is also highly specific for PI(3)P as a substrate. Furthermore, the MTM-related phosphatases MTMR1, MTMR3, and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this substrate is common to all myotubularin family enzymes. A direct comparison of the lipid phosphatase activities of recombinant myotubularin and MTMR2 demonstrates that their enzymatic properties are indistinguishable, indicating that the lack of functional redundancy between these proteins is likely to be due to factors other than the utilization of different physiologic substrates. To this end, we have analyzed myotubularin and MTMR2 transcripts during induced differentiation of cultured murine C2C12 myoblasts and find that their expression is divergently regulated. In addition, myotubularin and MTMR2 enhanced green fluorescent protein fusion proteins exhibit overlapping but distinct patterns of subcellular localization. Finally, we provide evidence that myotubularin, but not MTMR2, can modulate the levels of endosomal PI(3)P. From these data, we conclude that the developmental expression and subcellular localization of myotubularin and MTMR2 are differentially regulated, resulting in their utilization of specific cellular pools of PI(3)P. myotubularin myotubularin-related protein-tyrosine phosphatase phosphatidylinositol 3-phosphate Fab1/YOTB/Vac1p/EEA1 Charcot-Marie-Tooth enhanced green fluorescent protein glutathione S-transferase phosphate-buffered saline Myotubularin (MTM1)1 is a dual specificity protein-tyrosine phosphatase (PTP)-like enzyme that is mutated in X-linked myotubular myopathy, a severe congenital disorder in which muscle cell development is compromised (1Spiro A.J. Shy G.M. Gonatas N.K. Arch. Neurol. 1966; 14: 1-14Crossref PubMed Scopus (293) Google Scholar, 2Laporte J. Hu L.J. Kretz C. Mandel J.-L Kioschis P. Coy J.F. Klauck S.M. Poutska A. Dahl N. Nat. Genet. 1996; 13: 175-182Crossref PubMed Scopus (517) Google Scholar, 3Laporte J. Biancalana V. Tanner S.M. Kress W. Schneider V. Wallgren-Petterson C. Herger F. Buj-Bello A. Blondeau F. Liechti-Gallati S. Mandel J.-L. Hum. Mutat. 2000; 15: 393-409Crossref PubMed Scopus (180) Google Scholar). Myogenesis in affected individuals is arrested at a late stage of differentiation/maturation following myotube formation, and the muscle cells have characteristic large centrally located nuclei (1Spiro A.J. Shy G.M. Gonatas N.K. Arch. Neurol. 1966; 14: 1-14Crossref PubMed Scopus (293) Google Scholar). The MTM1 protein is the first characterized member of one of the largest families of dual specificity PTPs yet identified (reviewed in Refs. 4Wishart M.J. Taylor G.S. Slama J.T. Dixon J.E. Curr. Opin. Cell Biol. 2001; 13: 172-181Crossref PubMed Scopus (61) Google Scholar and 5Laporte J. Blondeau F. Buj-Bello A. Mandel J.-L. Trends Genet. 2001; 17: 221-228Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The MTM family includes at least eight putative catalytically active proteins as well as four forms that are predicted to be enzymatically inactive (4Wishart M.J. Taylor G.S. Slama J.T. Dixon J.E. Curr. Opin. Cell Biol. 2001; 13: 172-181Crossref PubMed Scopus (61) Google Scholar, 5Laporte J. Blondeau F. Buj-Bello A. Mandel J.-L. Trends Genet. 2001; 17: 221-228Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 6Appel S. Reichwald K. Zimmermann W. Reis A. Rosenthal A. Hennies H.C. Genomics. 2001; 75: 6-8Crossref PubMed Scopus (10) Google Scholar, 7Nandurkar H.H. Caldwell K.K. Whisstock J.C. Layton M.J. Gaudet E.A. Norris F.A. Majerus P.W. Mitchell C.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9499-9504Crossref PubMed Scopus (41) Google Scholar). The inactive MTM proteins contain substitutions at specific residues that are required for catalysis by PTP superfamily enzymes and may function as interaction modules (4Wishart M.J. Taylor G.S. Slama J.T. Dixon J.E. Curr. Opin. Cell Biol. 2001; 13: 172-181Crossref PubMed Scopus (61) Google Scholar, 5Laporte J. Blondeau F. Buj-Bello A. Mandel J.-L. Trends Genet. 2001; 17: 221-228Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 6Appel S. Reichwald K. Zimmermann W. Reis A. Rosenthal A. Hennies H.C. Genomics. 2001; 75: 6-8Crossref PubMed Scopus (10) Google Scholar, 7Nandurkar H.H. Caldwell K.K. Whisstock J.C. Layton M.J. Gaudet E.A. Norris F.A. Majerus P.W. Mitchell C.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9499-9504Crossref PubMed Scopus (41) Google Scholar, 8Wishart M.J. Dixon J.E. Trends Biochem. Sci. 1998; 23: 301-306Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Phylogenetic analysis of MTM family proteins indicates that they can be further divided into at least four distinct subgroups, which include the catalytically active MTM1/MTMR1/MTMR2, MTMR3/MTMR4, and MTMR6/MTMR7/MTMR8 enzymes, as well as the SBF1/LIP-STYX/MTMR10/3-PAP inactive forms (4Wishart M.J. Taylor G.S. Slama J.T. Dixon J.E. Curr. Opin. Cell Biol. 2001; 13: 172-181Crossref PubMed Scopus (61) Google Scholar, 5Laporte J. Blondeau F. Buj-Bello A. Mandel J.-L. Trends Genet. 2001; 17: 221-228Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 6Appel S. Reichwald K. Zimmermann W. Reis A. Rosenthal A. Hennies H.C. Genomics. 2001; 75: 6-8Crossref PubMed Scopus (10) Google Scholar, 7Nandurkar H.H. Caldwell K.K. Whisstock J.C. Layton M.J. Gaudet E.A. Norris F.A. Majerus P.W. Mitchell C.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9499-9504Crossref PubMed Scopus (41) Google Scholar). Our laboratory and others have previously shown that myotubularin specifically dephosphorylates the D3 position of the inositol lipid, phosphatidylinositol 3-phosphate (PI(3)P) (9Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (279) Google Scholar, 10Blondeau F. Laporte J. Bodin S. Superti-Furga G. Payrastre B. Mandel J.-L. Hum. Mol. Genet. 2000; 9: 2223-2229Crossref PubMed Scopus (215) Google Scholar). Recent reports have also shown that MTMR3 and MTMR4 (also referred to as FYVE-DSP1 and FYVE-DSP2, respectively) can utilize PI(3)P as a substrate (11Zhao R. Qi Y. Chen J. Zhao Z.J. Exp. Cell Res. 2001; 265: 329-338Crossref PubMed Scopus (45) Google Scholar, 12Walker, D. M., Urbe, S., Dove, S. K., Tenza, D., Raposo, G., and Clague, M. J. Curr. Biol., 11, 1600–1605Google Scholar). Together, these findings suggest that all of the active MTM family members may function to regulate cellular PI(3)P levels. Although it is well established that PI(3)P can serve as a targeting motif for membrane-trafficking and signaling proteins that contain lipid binding modules such as FYVE, pleckstrin homology, and Phox homology domains (13Gaullier J.-M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Crossref PubMed Scopus (446) Google Scholar, 14Patki V. Lawe D.C. Corvera S. Virbasius J.V. Chawla A. Nature. 1998; 394: 433-434Crossref PubMed Scopus (251) Google Scholar, 15Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 16Dowler S. Currie R.A. Campbell D.G. Deak M. Kular G. Downes C.P. Alessi D.R. Biochem. J. 2000; 351: 19-31Crossref PubMed Scopus (479) Google Scholar, 17Cheever M.L. Sato T.K. de Beer T. Kutateladze T. Emr S.D. Overduin M. Nat. Cell Biol. 2001; 3: 613-618Crossref PubMed Scopus (316) Google Scholar, 18Xu Y. Hortsman H. Seet L. Wong S.H. Hong W. Nat. Cell Biol. 2001; 3: 658-666Crossref PubMed Scopus (240) Google Scholar, 19Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (501) Google Scholar, 20Ellson C.D. Gobert-Grosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.-Y. Holmes A.B. Gaffney P.R.J. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Crossref PubMed Scopus (362) Google Scholar), the precise physiologic roles of MTM family phosphatases as regulators of PI(3)P are unclear. In addition to MTM1 mutations identified in X-linked myotubular myopathy, a second MTM-related gene has been associated with a human genetic disease. Mutations in theMTMR2 gene are causative for the neurodegenerative disorder, type 4B Charcot-Marie-Tooth (CMT) syndrome, which is a hereditary demyelinating peripheral neuropathy that results from improper Schwann cell development (21Bolino A. Brancolini V. Bono F. Bruni A. Gambardella A. Romeo G. Quattrone A. Devoto M. Hum. Mol. Genet. 1996; 5: 1051-1054Crossref PubMed Scopus (113) Google Scholar, 22Bolino A. Muglia M. Conforti F.L. LeGuern E. Salih M.A. Georgiou D.M. Christodoulou K. Hausmanowa,-Petrusewicz I. Mandich P. Schenone A. Gambardella A. Bono F. Quattrone A. Devoto M. Monaco A.P. Nat. Genet. 2000; 25: 17-19Crossref PubMed Scopus (423) Google Scholar, 23Houlden H. King R.H. Wood N.W. Thomas P.K. Reilly M.M. Brain. 2001; 124: 907-915Crossref PubMed Scopus (79) Google Scholar). Patients with type 4B CMT exhibit abnormal focally folded myelin in the neural sheath (21Bolino A. Brancolini V. Bono F. Bruni A. Gambardella A. Romeo G. Quattrone A. Devoto M. Hum. Mol. Genet. 1996; 5: 1051-1054Crossref PubMed Scopus (113) Google Scholar). The difference in the pathologies of myotubular myopathy and type 4B CMT are particularly intriguing in light of the fact that MTM1 and MTMR2 are both expressed in adult skeletal muscle and neuronal tissues and are highly similar (64% identity, 76% similarity) (24Laporte J. Blondeau F. Buj-Bello A. Tentler D. Kretz C. Dahl N. Mandel J.-L. Hum. Mol. Genet. 1998; 7: 1703-1712Crossref PubMed Scopus (99) Google Scholar). Although their specific physiologic roles are not known, it is clear from the different pathologies manifested in myotubular myopathy and CMT disease that MTM1 and MTMR2 are not functionally redundant. The lack of functional overlap between these proteins may have several possible explanations. Although they are highly similar at the protein level, MTM1 and MTMR2 may have distinct substrate preferences. Alternatively, although they are expressed in both adult skeletal muscle and neuronal tissues, MTM1 and MTMR2 may play important roles during specific developmental stages and thus be expressed differentially during maturation of specific tissues. Finally, they may both act on PI(3)P but be localized to distinct subcellular compartments, thus regulating different cellular pools of this lipid. Consistent with this notion, a recent study has demonstrated that PI(3)P can be detected at multiple discrete sites within internal vesicular structures, including endosomes and endosomal carrier vesicles as well as the nucleolus (25Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (863) Google Scholar). In the current study, we have undertaken the enzymatic characterization of MTM family phosphatases to determine whether utilization of the inositol lipid, PI(3)P, as a substrate is common among these enzymes. As a first step toward understanding the molecular basis of MTM function in the human genetic disorders, myotubular myopathy and type 4B CMT, we have also conducted a detailed comparison of the enzymatic properties of recombinant MTM1 and MTMR2. Our results demonstrate that like MTM1, MTMR2 can act on PI(3)P, providing a direct link between inositol lipid regulation and type 4B CMT. In addition, we have analyzed the subcellular localization and expression of MTM1 and MTMR2 during myogenic differentiation to determine whether these factors might contribute to their functional regulation. Finally, we present evidence that MTM1 and MTMR2 are likely to act on distinct cellular pools of PI(3)P. COS-1 and C2C12 cells were maintained at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. C2C12 cell differentiation was induced in Dulbecco's modified Eagle's medium containing 5% horse serum, 50 units/ml penicillin, and 50 μg/ml streptomycin for up to 5 days at 37 °C with 5% CO2. COS-1 cells were transiently transfected using Fugene 6 reagent (Roche Molecular Biochemicals) according to the manufacturer's protocol. The completeMTM1 open reading frame (1812 nucleotides) used in constructs described herein corresponds to GenBankTMaccession number NM_000252. The complete MTMR1 open reading frame (2022 nucleotides) was amplified by PCR using a mixture of two partial cDNA fragments as a template. The 5′-region ofMTMR1 (nucleotides 1–242) was amplified by PCR using human IMAGE expressed sequence tag clone 2125570 (Research Genetics/Invitrogen, Huntsville, AL) as a template. The 3′-region ofMTMR1 (nucleotides 141–1991 of GenBankTMaccession number AJ224979) was amplified from human skeletal muscle cDNA (CLONTECH, Palo Alto, CA). Equal amounts of gel-purified 5′- and 3′-cDNA fragments were then used as a template to amplify the complete MTMR1 open reading frame. The complete open reading frames of MTMR2 (1932 nucleotides) and MTMR3 (3597 nucleotides) were amplified by PCR from plasmids containing MTMR2 or MTMR3 cDNAs (KIAA1073 and KIAA0371 cDNA clones, respectively) obtained from the Kazusa DNA Research Institute (26Kikuno R. Nagase T. Suyama M. Waki M. Hirosawa M. Ohara O. Nucleic Acids Res. 2000; 28: 331-332Crossref PubMed Scopus (42) Google Scholar). The open reading frame ofMTMR6 (1866 nucleotides) corresponding to GenBankTM accession number AF406619 was amplified by PCR using human skeletal muscle cDNA as a template (CLONTECH). Vectors for the expression of bacterial recombinant His-tagged MTM1, MTMR1, MTMR2, and MTMR6 were created using the pET21a vector (Stratagene, La Jolla, CA). The pET-MTM1 expression construct has been previously described (27Taylor G.S. Dixon J.E. Anal. Biochem. 2001; 295: 122-126Crossref PubMed Scopus (28) Google Scholar). The pET-MTMR1 vector was created by inserting a DNA fragment containing the MTMR1 open reading frame without a stop codon into the 5′-NheI and 3′-XhoI sites of pET21a in-frame with the six-histidine tag. The pET-MTMR2 construct was created by inserting a DNA fragment containing the MTMR2 open reading frame without a stop codon into the 5′-NheI and 3′-NotI sites of pET21a in-frame with the six-histidine tag. The pET-MTMR6 expression construct was created by inserting a DNA fragment containing the MTMR6open reading frame without a stop codon into the 5′-BamHI and 3′-HindIII sites of pET21a in-frame with the six-histidine tag. Vectors for the expression of N-terminally FLAG-tagged MTM1 wild type and C375S mutant proteins in mammalian cells have been previously described (9Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (279) Google Scholar). Mammalian expression vectors for N-terminally FLAG-tagged MTMR1, MTMR2, MTMR3, and MTMR6 proteins were created by inserting cDNA fragments containing the complete open reading frames encoding each of these proteins into the 5′-BamHI/3′-NotI, 5′-NheI/3′-KpnI, 5′-NheI/3′-XbaI, and 5′-BamHI/3′-XbaI, respectively, of pCDNA3.1-NF (9Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (279) Google Scholar). The EGFP-MTM1 mammalian expression vector has been previously described (9Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (279) Google Scholar). A cDNA fragment containing the complete open reading frame ofMTMR2 was inserted into the 5′-BglII and 3′-KpnI sites of the pEGFP-C1 vector (CLONTECH) to create pEGFP-MTMR2. A vector for bacterial expression of tandem FYVE domains from the murine hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) protein fused to GST (GST-2×FYVE; see Ref. 25Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (863) Google Scholar) was the generous gift of Dr. Kathleen Collins (University of Michigan). Bacterial recombinant MTM1 and MTMR2 C-terminally His-tagged fusion proteins used for phosphatase assays were expressed in Escherichia coliBL21(DE3) Codon Plus cells (Stratagene) and purified using Ni2+-agarose affinity resin as previously described forSacIp (28Maehama T. Taylor G.S. Slama J.T. Dixon J.E. Anal. Biochem. 2000; 279: 248-250Crossref PubMed Scopus (99) Google Scholar). FLAG-tagged MTM proteins used for phosphatase assays were expressed in COS-1 cells and purified using anti-FLAG M2 affinity resin (Sigma) as previously described for FLAG-tagged MTM1 wild-type and C375S proteins (27Taylor G.S. Dixon J.E. Anal. Biochem. 2001; 295: 122-126Crossref PubMed Scopus (28) Google Scholar). Recombinant GST-2×FYVE protein was expressed in Escherichia coli DH5α cells and purified over glutathione-agarose affinity resin. Briefly, cells harboring the pGEX-2×FYVE plasmid were grown in 2× YT medium containing 100 μg/ml ampicillin to an A600 of 0.7, and then protein expression was induced by the addition of isopropyl β-d-thiogalactopyranoside (0.5 mm) for 2 h at 37 °C. The cells were harvested by centrifugation and resuspended in PBS (pH 7.4) containing 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml (each) aprotinin, leupeptin, and pepstatin (30 ml of lysis buffer per liter of cultured cells). The cells were disrupted by sonication, and Triton X-100 was added to 0.5% (v/v). The crude lysate was centrifuged for 30 min at 18,000 × g to remove unbroken cells and insoluble debris. The soluble fraction was incubated with glutathione-agarose affinity resin (1 ml of 50% slurry in PBS/liter of culture) for 2 h at 4 °C. The resin was washed three times for 5 min each with lysis buffer containing 0.5% Triton X-100, followed by two 5-min washes in lysis buffer without detergent. The GST-2×FYVE fusion protein was eluted from the resin for 2 × 30 min with 0.5 ml of lysis buffer containing 25 mm reduced glutathione (pH 7.4) and filtered through a 0.2-μm syringe filter. Free glutathione was removed by gel filtration chromatography in PBS using a Superdex 200 column and FPLC chromatography system (Amersham Biosciences, Inc.). The GST-2×FYVE fusion protein was biotinylated using the Biotin-Tag microbiotinylation kit as per the manufacturer's protocol (Sigma-Aldrich). The unreacted biotinylation reagent was removed using a PD-10 desalting column (Bio-Rad). All phosphoinositide and soluble inositol phosphate substrates used in this work were obtained from Echelon Research Laboratories (Salt Lake City, UT). Phosphatase assays using FLAG-tagged MTM proteins immunoprecipitated from COS-1 cells were carried out on anti-FLAG M2 affinity resin in reaction buffer containing 1.5 μg of di-C6-NBD6-phosphatidylinositol 3-phosphate (Echelon) as described (27Taylor G.S. Dixon J.E. Anal. Biochem. 2001; 295: 122-126Crossref PubMed Scopus (28) Google Scholar). Samples were separated by thin layer chromatography, and the reaction products were visualized under UV light (27Taylor G.S. Dixon J.E. Anal. Biochem. 2001; 295: 122-126Crossref PubMed Scopus (28) Google Scholar). Phosphatase assays with bacterial recombinant MTM1, MTMR1, MTMR2, and MTMR6 using synthetic di-C8-phosphoinositide or soluble inositol phosphate substrates were carried out at 30 °C, and phosphate release was determined using a malachite green-based assay system for inorganic phosphate as described (28Maehama T. Taylor G.S. Slama J.T. Dixon J.E. Anal. Biochem. 2000; 279: 248-250Crossref PubMed Scopus (99) Google Scholar). The artificial protein phosphatase substrates myelin basic protein and casein were phosphorylated with [γ-32P]ATP on tyrosyl or seryl/threonyl residues and used as substrates for recombinant MTM1 and MTMR2 as previously described (9Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (279) Google Scholar). Total RNA was isolated from C2C12 cells with TRIzol reagent as recommended by the manufacturer (Invitrogen). RNA samples (15 μg) were then electrophoresed through a 1% agarose-formaldelyde gel and transferred to a nylon membrane. The blots were hybridized with digoxigenin-labeled probes, and mRNAs corresponding to murine MTM1 and MTMR2 were detected using a digoxigenin chemiluminescence detection kit (Roche Molecular Biochemicals). A murine MTM1 cDNA probe corresponding to the 3′-untranslated region (283 bp) was amplified by PCR using expressed sequence tag clone AW911959 (Research Genetics/Invitrogen) as a template. A cDNA probe corresponding to the murine MTMR2 5′-region (nucleotides −5 to +381) was amplified by PCR using expressed sequence tag clone AI561725 (Research Genetics/Invitrogen) as a template. COS-1 cells were seeded on two-chamber slides (21 × 21 mm) at a density of 5 × 104cells/well in Dulbecco's modified Eagle's medium and cultured as described above. After attachment (∼8 to 12 h), the cells were transiently transfected using Fugene 6 reagent as described above. At 30 h post-transfection, the cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Coverslips were mounted on the slides using ProLong Antifade mounting medium (Molecular Probes, Eugene, OR). For analyses with biotinylated GST-2×FYVE as a probe for PI(3)P, a modified form of a protocol developed in the laboratory of Dr. Harald Stenmark at the Department of Biochemistry, Institute for Cancer Research, Norwegian Radium Hospital, Oslo, Norway, was used. 2H. Stenmark, personal communication.Briefly, COS-1 cells were cultured and transfected on chambered microscope slides as described above. At 24 h post-transfection, the cells were washed and fixed overnight at 4 °C in 4% paraformaldehyde in PBS, which permeabilizes most of the cells. The cells were washed once in PBS, then the free aldehyde groups were quenched by incubation with PBS containing 50 mmNH4Cl for 15 min at room temperature. All subsequent steps were carried out at room temperature. The slides were incubated for 1 h with 10% fetal calf serum in PBS as a blocking agent. Next, the slides were incubated with 3% fetal calf serum in PBS containing 50 μg/ml biotinylated GST-2×FYVE for 30 min. The slides were washed for 3 × 5 min with PBS. The slides were then incubated for 30 min with 1 μg/ml Cy3-conjugated streptavidin (Sigma-Aldrich) in PBS containing 3% fetal calf serum. Finally, the slides were washed three times for 5 min each with PBS and mounted using ProLong mounting medium. Fluorescence analyses were performed by conventional fluorescence microscopy or by confocal microscopy (Zeiss LSM 510) as indicated. The association of MTM1 and MTMR2 with the respective genetic disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome is of significant interest because although they are highly similar proteins and exhibit overlapping expression patterns in human tissues, they do not appear to be functionally redundant (2Laporte J. Hu L.J. Kretz C. Mandel J.-L Kioschis P. Coy J.F. Klauck S.M. Poutska A. Dahl N. Nat. Genet. 1996; 13: 175-182Crossref PubMed Scopus (517) Google Scholar, 21Bolino A. Brancolini V. Bono F. Bruni A. Gambardella A. Romeo G. Quattrone A. Devoto M. Hum. Mol. Genet. 1996; 5: 1051-1054Crossref PubMed Scopus (113) Google Scholar, 22Bolino A. Muglia M. Conforti F.L. LeGuern E. Salih M.A. Georgiou D.M. Christodoulou K. Hausmanowa,-Petrusewicz I. Mandich P. Schenone A. Gambardella A. Bono F. Quattrone A. Devoto M. Monaco A.P. Nat. Genet. 2000; 25: 17-19Crossref PubMed Scopus (423) Google Scholar, 23Houlden H. King R.H. Wood N.W. Thomas P.K. Reilly M.M. Brain. 2001; 124: 907-915Crossref PubMed Scopus (79) Google Scholar). As a first step toward understanding the functional differences between MTM1 and MTMR2, we analyzed their enzymatic properties to determine whether they possessed similar activity and/or substrate specificity. Recombinant MTM1 and MTMR2 fusion proteins were expressed and purified as described under “Experimental Procedures,” and their phosphatase activity was tested toward a panel of substrates including phosphoinositides, soluble inositol phosphates, and radiolabeled artificial protein substrates. As shown in Table I, the activity of MTMR2 was indistinguishable from that of MTM1 over the panel of substrates tested. Both enzymes dephosphorylated PI(3)P >50-fold more efficiently than any other substrate, demonstrating that they are highly specific for this lipid (Table I). Although MTM1 and MTMR2 were able to dephosphorylate the PI(3)P soluble headgroup analog, inositol 1,3-bisphosphate, their activity with this substrate was 10-fold lower than with PI(3)P, indicating a strong preference for the lipid as a substrate. Neither enzyme exhibited significant activity toward artificial protein substrates phosphorylated on tyrosyl or seryl/threonyl residues, confirming that they are extremely poor protein phosphatases. Most importantly, because the truncatingMTMR2 mutations first associated with type 4B CMT syndrome would cause loss of MTMR2 phosphatase activity, our findings suggest that this disorder, like myotubular myopathy, results from the failure to regulate cellular PI(3)P levels (9Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (279) Google Scholar, 22Bolino A. Muglia M. Conforti F.L. LeGuern E. Salih M.A. Georgiou D.M. Christodoulou K. Hausmanowa,-Petrusewicz I. Mandich P. Schenone A. Gambardella A. Bono F. Quattrone A. Devoto M. Monaco A.P. Nat. Genet. 2000; 25: 17-19Crossref PubMed Scopus (423) Google Scholar). Furthermore, these data indicate that the lack of functional redundancy between MTM1 and MTMR2 is not due to differences in their catalytic properties or substrate specificity.Table IPhosphatase activity of recombinant myotubularin and MTMR2Substrate1-aPI, phosphatidylinositol; P2, bisphosphate; P3, trisphosphate; P4, tetraphosphate; MBP, myelin basic protein.ConcentrationSpecific activityMTM1-H6MTMR2-H6μmmol min−1 mol−1p-Nitrophenol phosphate50,0004459PI(3)P5022262281PI(4)P501016PI(5)P504648PI(3,4)P2501301-bLimit of detection in these assays was 10 pmol of phosphate released.PI(3,5)P25001-bLimit of detection in these assays was 10 pmol of phosphate released.01-bLimit of detection in these assays was 10 pmol of phosphate released.PI(4,5)P25001-bLimit of detection in these assays was 10 pmol of phosphate released.01-bLimit of detection in these assays was 10 pmol of phosphate released.PI(3,4,5)P35001-bLimit of detection in these assays was 10 pmol of phosphate released.10Ins(4)P5001-bLimit of detection in these assays was 10 pmol of phosphate released.01-bLimit of detection in these assays was 10 pmol of phosphate released.Ins(1,3)P250206232Ins(1,4)P25001-bLimit of detection in these assays was 10 pmol of phosphate released.2Ins(1,3,4)P35034Ins(1,4,5)P35001-bLimit of detection in these assays was 10 pmol of phosphate released.01-bLimit of detection in these assays was 10 pmol of phosphate released.Ins(1,3,4,5)P45022Tyr(P)-MBP5.00.0180.015Tyr(P)-casein5.00.1060.055Ser(P)/Tyr(P)-MBP5.00.00040.0009Ser(P)-casein5.00.00020.0006Phosphatase assays using purified recombinant MTM1 and MTMR2 proteins were carried out at 30 °C with the indicated amount of substrate as described under “Experimental Procedures.” The specific activities shown are expressed as mol of phosphate released/min/mol of enzyme and represent the mean of triplicate determinations. S.E. of each determination was less than 5%.1-a PI, phosphatidylinositol; P2, bisphosphate; P3, trisphosphate; P4" @default.
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• W2007826893 title "Myotubularin and MTMR2, Phosphatidylinositol 3-Phosphatases Mutated in Myotubular Myopathy and Type 4B Charcot-Marie-Tooth Disease" @default.
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