Human Molecular Genetics, 2000, Vol. 9, No. 15 2223-2229
© 2000 Oxford University Press
Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP 163, 67404 Illkirch Cedex, CU de Strasbourg, France, 1INSERM U326, CHU Purpan, 31059 Toulouse Cedex France and 2EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Received 24 July 2000; Accepted 7 August 2000.
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ABSTRACT |
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Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres. The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus. It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot–Marie–Tooth disease. Myotubularin shows a dual specificity protein phosphatase activity in vitro. We have performed an in vivo test of tyrosine phosphatase activity in Schizosaccharomyces pombe, indicating that myotubularin does not have a broad specificity tyrosine phosphatase activity. Expression of active human myotubularin inhibited growth of S.pombe and induced a vacuolar phenotype similar to that of mutants of the vacuolar protein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylinositol 3-kinase (PI3K). In S.pombe cells deleted for the endogenous MTM homologous gene, expression of human myotubularin decreased the level of phosphatidylinositol 3-phosphate (PI3P). We have created a substrate trap mutant which shows relocalization to plasma membrane projections (spikes) in HeLa cells and was inactive in the S.pombe assay. This mutant, but not the wild-type or a phosphatase site mutant, was able to immunoprecipitate a VPS34 kinase activity. Wild-type myotubularin was also able to directly dephosphorylate PI3P and PI4P in vitro. Myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.
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INTRODUCTION |
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X-linked recessive myotubular myopathy (XLMTM) is a very severe congenital muscular disease characterized by hypotonia and generalized muscle weakness in newborn males. Most patients die within the first months of life. Characteristic muscle histopathology consists of small rounded muscle cells with centrally located nuclei resembling fetal myotubes. It has been suggested that the disorder results either from an arrest in the normal development of muscle fibres or from a defect in the structural organization of myofibres. There is no abnormality in myoblast fusion. The locus responsible for the disease (MTM1) was mapped to proximal Xq28 and the MTM1 gene was identified by positional cloning (1). It encodes a protein, myotubularin, with a putative dual-specificity protein phosphatase domain [PTP/DSP (2,3)]. Myotubularin is highly conserved through evolution and eight homologous human genes were found, which define a large family of phosphatases from yeasts to human (2). In this family, one member has been recently found mutated in a recessive form of Charcot–Marie–Tooth disease [hMTMR2 (4)].
Understanding myotubularin activity should thus shed light on the function of the members of the MTM gene family already linked to two different diseases, a myopathy and a demyelinating neuropathy. In this study, we have investigated the phosphatase activity and substrates of myotubularin in vitro and in vivo. We have found that in a yeast expression model the human MTM1 and the endogenous Schizosaccharomyces pombe MTM act on the phosphatidylinositol 3-kinase [VPS34, the PI3K protein (5)] pathway and down-regulate the phosphatidylinositol 3-phosphate (PI3P) level.
We provide evidence that myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.
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RESULTS |
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The putative tyrosine phosphatase activity of human myotubularin (hMTM1) was investigated in vivo in S.pombe (6). When we co-expressed c-Src (which heavily phosphorylates endogenous proteins on tyrosine) and wild-type hMTM1 (hMTM1-WT), we were unable to detect any dephosphorylation of tyrosine containing proteins, by western blotting of one and two-dimensional polyacrylamide gels (Fig. 1a, and data not shown). In contrast, a strong reduction of tyrosine phosphorylation was observed when c-Scr was co-expressed with the PEST tyrosine phosphatase (PTP-PEST) and the T cell tyrosine phosphatase (TC-PTP) (Fig. 1a) (6). Moreover, transfection of wild-type myotubularin into 3T3 cells expressing the constitutively activated Src mutant Y527F (7) did not revert the transformed phenotype (ascertained by cell morphology) and did not affect tyrosine phosphorylation (data not shown). We conclude either that myotubularin is not a tyrosine phosphatase or that it may have a much more restricted substrate specificity than PTP-PEST or TC-PTP.
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However, S.pombe cells expressing a high level of hMTM1-WT exhibited a phenotype of decrease in growth rate (Fig. 2a) and the presence of large vacuoles (Fig. 2c). This phenotype was not induced when expressing a myotubularin mutated at the catalytic cysteine in the phosphatase active site (hMTM1-C375S). Doubling time was 13.4 h for cells expressing hMTM1-WT, against 4.4 h with hMTM1-C375S, and 3.8 h for mock-transformed cells. Cells expressing a high level of the two broad-specificity PTPs (PTP-PEST and TC-PTP) did not exhibit the vacuolar phenotype (data not shown) or decreased cell growth (6). The vacuoles observed in cells expressing human myotubularin accumulate carboxy fluorescein di-acetate (CFDA) (Fig. 2c), a marker of acidic yeast vacuoles (8). These big vacuoles are likely to be due to accumulation and fusion of smaller ones.
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This phenotype is very similar to that observed in S.pombe mutants with a loss of function of proteins belonging to the vacuolar protein sorting (VPS) class (5) (Fig. 2c). Among them, VPS34 is a PI3K which phosphorylates phosphatidylinositol into PI3P. To analyse the effect of myotubularin on phosphoinositides in S.pombe, we replaced the endogenous MTM homologue (SpMTM; GenBank accession no. Z98974) by a neomycine resistance gene cassette (9). The two independent
SpMTM mutants did not show any obvious phenotype under standard growth conditions. However, these two strains exhibited two additional bands at the level of PI3P and possibly of PI3,5P2 (arrowhead and asterisk in Fig. 3a). Indeed, the upper band was unambiguously identified as PI3P by HPLC (Fig. 3b).
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These bands disappeared or decreased in intensity in
SpMTM strains expressing hMTM1-WT, whereas inactive mutants hMTM1-D278A (see below) or hMTM1-C375S did not have this effect (Fig. 3a, compare lanes 3 and 4, and data not shown). These results suggest that both the endogenous SpMTM and active human myotubularin down-regulate production of PI3P or enhance its degradation. As expression of human myotubularin leads to a phenotype similar to a loss of VPS34 (PI3K), we investigated whether VPS34 might be a substrate of myotubularin. For this, we took advantage of the dominant negative substrate trapping strategy that was successful for other tyrosine phosphatases, i.e. changing the aspartate required for the release of the substrate into alanine (10). We individually mutated six aspartate residues conserved within the myotubularin family to alanine residues. Four mutants were unstable in HeLa cells, indicating that the mutation affects a structural aspartate (data not shown). hMTM1-D377A showed a cytoplasmic localization similar to that of hMTM1-WT and hMTM1-C375S proteins (Fig. 4a) whereas hMTM1-D278A delocalized to plasma membrane extensions (Fig. 4b–c). As shown by electron microscopy, hMTM1-D278A was highly concentrated over membrane microvilli but no membrane-associated signal was detected on the basal membrane in contact with the substratum (Fig. 4d–e). Thus, this suggests that a substrate of myotubularin is present in HeLa cells in phosphorylated form, at a precise location close to the plasma membrane.
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We expressed these two D
A mutants of human myotubularin in S.pombe. Human MTM1-D377A mutant inhibited cell growth and induced vacuole formation, whereas hMTM1-D278A mutant was inactive (data not shown). Aspartate D377 is adjacent to the catalytic cysteine within the PTP active site. Aspartate D278 is within a conserved region separated from the catalytic pocket by a region predicted to form a loose conformation, fitting in with the model of other PTPs (11). Moreover, unlike wild-type myotubularin, hMTM1-D278A produced in Baculovirus-infected SF9 cells, had no activity on paranitrophenyl phosphate synthetic substrate (data not shown).
We were able to detect a phosphatidylinositol kinase activity after immunoprecipitation with a myotubularin antibody of S.pombe protein extract containing the inactive hMTM1-D278A (Fig. 5a). The labelled phosphorylated product co-migrated with PI3P generated from immunoprecipitated mouse P110
PI3K (Fig. 5a, lane 5). No PI3K activity was detected when the active hMTM1-WT or the inactive hMTM1-C375S was immunoprecipitated (Fig. 5a, lanes 1–2), indicating that their interaction with VPS34 is either absent or rather transient, as expected for a phosphatase–substrate interaction. Moreover, we were able to co-immunoprecipitate the myotubularin substrate trap mutant hMTM1-D278A more efficiently than the wild-type using SpVPS34 antibody (data not shown).
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-32P]ATP to assay PI3K activity. The control was COS cells transfected with a myc-tagged mouse p110
0.4 (n = 2).We also investigated whether myotubularin might dephosphorylate directly PI3P or other phosphoinositides in vitro. Extracts of S.pombe expressing either wild-type human myotubularin or the D278A mutant were immunoprecipitated and incubated with 32P-labelled PI3P. Indeed, wild-type myotubularin was able to efficiently dephosphorylate PI3P, whereas the D278A mutant was inactive (Fig. 5b). We also observed that immunoprecipitated hMTM1 was able to hydrolyse PI4P to some extent. Therefore, in order to avoid any potential contaminant phosphatase activity that could be co-immunoprecipitated and to compare the specific activity using PI3P and PI4P as substrate, we have performed a phosphatase assay using recombinant myotubularin. In these conditions, we clearly measured a phosphatase activity against PI3P and to a lesser extent PI4P (95.4 nmol/min/mg for PI3P and 45.3 nmol/min/mg for PI4P), whereas the hMTM1-C375S mutant was totally inactive against these substrates (data not shown). Wild-type myotubularin also dephosphorylated to a lesser extent PI3,4P2 and very weakly PI4,5P2, whereas it had no significant activity on PI3,4,5P3 (Fig. 5b legend, and data not shown). However, results shown in Figure 3 suggest that in S.pombe the activity with PI3P is more significant than that with PI4P.
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DISCUSSION |
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In the yeast secretory pathway, several phosphorylated factors are known to be required. A Ser/Thr kinase (VPS15) is required to activate a PI3K (VPS34) and both proteins are phosphorylated in vivo in Saccharomyces cerevisiae (12,13). These two proteins are also present in Schizosaccharomyces pombe (5, and K. Takegawa, personal communication). Several mammalian PI3Ks are known to be phosphorylated for activation, including PI3K involved in insulin/IGF action (14). Our experiments in S.pombe indicated that both endogenous SpMTM or active human MTM1 decrease the level of phosphoinositides. This could be due either to a direct effect of the phosphatase on the phospholipid substrates, as was shown for the PTEN phosphatase (15), or to an effect on the regulation of their synthesis. Two highly conserved lysine residues that play an important role in the interaction of PTEN with negatively charged PI3K products (16,17) are absent in myotubularin. Two completely conserved aspartate residues would confer an acidic, rather than basic, character to the active site of myotubularin and homologous proteins. This could explain why hMTM1-WT preferentially dephosphorylates phosphoinositide monophosphate in vitro. The results of the substrate trap experiment strongly suggest that myotubularin is also able to dephosphorylate VPS34 and would thus down-regulate its PI3K activity. The action of myotubularin on PI3P levels may implicate two parallel pathways by acting both as a protein phosphatase on PI3K and a lipid phosphatase, preferentially on PI3P than PI4P in vivo. Such a dual activity has also been suggested for PTEN (18).
The subcellular relocalization of the substrate-trapping myotubularin mutant and the fact that myotubularin acts on the PI3K pathway suggest a role in relaying/regulating signals elicited by growth factors during myogenesis. Considering the critical role of the PI3K enzymes and of the insulin pathway (19), we will investigate the effect of hMTM1 in muscle maturation. It should be added that we always found wild-type myotubularin localized in the cytoplasm in transfection experiments performed on a large variety of cell lines (Fig. 4) (J. Laporte, manuscript in preparation), in contradiction with the nuclear localization reported for a tagged version of myotubularin and for Sbf1/MTMR5 (3).
The myotubularin gene family can be divided into four groups (2). The hMTM1 gene and two other human genes, including MTMR2 implicated in the CMT4B form of Charcot–Marie–Tooth disease [a demyelinating neuropathy (4)], belong to the same subgroup as a zebrafish and a Drosophila gene and are more distantly related to the other human genes (2). This conservation of subgroups over a long evolutionary time suggests that these phosphatases have acquired specific functions. Indeed, in two of the subgroups, additional protein domains related to phosphoinositides are present: the pleckstrin homology domain in MTMR5/Sbf1 (20) and the ring FYVE domain of MTMR3 (21). As most of the human genes in this family (including the disease-related MTM1 and MTMR2 genes) show a ubiquitous expression, the specificity should be in substrate recognition or regulatory interactions. Different myotubularin homologues might thus interact with different PI3Ks and/or act on different monophosphate lipid substrates. This study gives a new insight into the comprehension of the disease and regulation of the PI3K pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Cells, constructs and antibodies
3T3 srcY527F fibroblasts were provided by Dr H. Boeuf (IGBMC). The active site mutant (hMTM1-C375S) (2) and the different aspartate mutants (hMTM1-D278, D377, D380, D394, E410 and D443) were engineered by PCR-based mutagenesis from wild-type myotubularin cDNA (GenBank accession no. U46024). Wild-type human hMTM1 cDNA and the various point mutants were subcloned into a pNU vector [wild-type nmt1 promoter, ura4 gene containing expression vector (G. Superti-Furga, unpublished data)] for transformation of S.pombe, into the pCS2 expression vector (22) for transfection into HeLa and 3T3 cells and into the pAcSG His NT-B vector (Pharmingen, San Diego, CA) for production in the Baculovirus system. An enzymatic test on paranitrophenyl phosphate was performed as described (2). The VPS34-expressing plasmid was a gift from Dr D.B. DeWald (5). The constitutively active myc-tagged P110 was a gift from Dr A. Klippel (23). Mock-transfection and transformation were done with empty vector. The 1G6 monoclonal antibody was raised against an N-terminal peptide (amino acids 13–32) of human myotubularin which is not conserved within the S.pombe myotubularin. 4G10 anti-phosphotyrosine antibody was from UBI (Lake Placid, NY), 9E10 anti-myc from the ATCC [CRL-1729; Rockville, MD (24)], 2-17 anti-src from G. Superti-Furga (6) and SpVPS34 antibody from K. Takegawa (5).
Transfection and subcellular localization
HeLa and 3T3 srcY527F cells were transfected with either the calcium phosphate method or Ex-Gen 500 (Euromedex, Souffelweyersheim, France). Cells were fixed in 4% paraformaldehyde 36 h after transfection and permeabilized in phosphate-buffered saline with 0.3% Triton X-100. Subcellular localization was assessed using the 1G6 antibody (1:1000 dilution) followed by Cy3-conjugated goat anti-mouse antibody (1:250; Jackson Immunoresearch, West Grove, PA). Alternatively, transfected cells were fixed without permeabilization, incubated with the 1G6 antibody followed by a goat anti-mouse IgG labelled with ultra small gold (1:200; Aurion, Wageningen, The Netherlands) and the immunoreactivity was revealed with the G-Rent gold signal enhancement system (Aurion). Cells were then subjected to conventional electron microscopy.
Yeast work
The wild-type S.pombe strain used was SP813 (h–n leu1-32 ura4-D18 ade6-210). Deletion of myotubularin was obtained by constructing a kanamycin vector (9) flanked by two 200 bp regions of S.pombe myotubularin (GenBank accession no. Z98974) amplified by PCR (primer sequences were 5'-AGAAAAAAGCGGCCGCTAGCCTTGATAACCGCAATGAT-3', 5'-GCTCTAGAGCAACCCTCGTACTATCAAGTA-3' and 5'-GGGACTATTTCCTTTCTCGG-3', 5'-CCCTTCGAAATGTTTCATAAAGACGGAGT-3'). Selection of deleted strains was performed on YEA (yeast extract adenine) medium containing geneticin (100 mg/l). Transformation of yeast, medium, phosphate labelling, CFDA accumulation and growth curve were as described previously (6,8,25,26).
PI3K and PI3P phosphatase in vitro assays
Yeast cells were lysed using glass beads in lysis buffer (50 mM Tris pH 7.8, 1% NP40, 150 mM NaCl). Protein extract was pre-cleared before immunoprecipitation with the different antibodies. Protein G–Sepharose beads were washed once in lysis buffer and twice in 25 mM Tris pH 7.8, 500 mM NaCl, then once in the kinase assay buffer or in phosphatase assay buffer. Beads were then tested for a phosphatidylinositol kinase activity in vitro. The assay was carried out in 50 µl of 10 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 100 µM adenosine (a potent inhibitor of most PI4Ks), 10 µg of phosphatidylinositol, 0.1 mM ATP, 20 mM MgCl2, 6 µCi [32P]ATP, H2O at 25°C for 15 min. Reaction was stopped by adding CHCl3:CH3OH (v/v) and lipids were extracted, separated by thin layer chromatography (TLC) using freshly prepared CHCl3:CH3OH:NH4OH 4N (45:30:10, v/v) and visualized by autoradiography.
The phosphoinositide phosphatase activity was measured as follows. [32P]D3-phosphoinositides were produced and purified as indicated previously (27), and [32P]PI4P and [32P]PI4,5P2 were TLC-purified from 32P-labelled human blood platelets. TLC-purified [32P]phosphoinositides and phosphatidylserine as a carrier lipid, were dried under a stream of nitrogen and suspended in 30 µl of 20 mM MES pH 6.5, 0.6% octylglucoside, 2.5 mM EDTA, 100 mM KCl. Immunopurified wild-type MTM1 was suspended in 30 µl of the same buffer lacking octylglucoside and the reaction was immediately started by adding 30 µl of lipids, at 37°C for 30 min under gentle shaking. The phosphatase assay carried out with recombinant myotubularin (dialysed overnight at 4°C in the phosphatase buffer) was performed on labelled PI3P and PI4P whose concentrations were made up to 50 µM with dipalmitoyl PI3P and 1-stearoyl 2-arachidonoyl PI4P, respectively. The reaction mixture contained 
15 µM phosphoinositide and 160 µM phosphatidylserine. The reaction was stopped by adding CHCl3:CH3OH (v/v) and lipids were extracted, separated by TLC using CHCl3:CH3COCH3:CH3OH:CH3COOH:H2O [80:30:26:24:14 (v/v)] (28) and visualized by a PhosphorImager 445 SI (Molecular Dynamics, Sunnyvale, CA).
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ACKNOWLEDGEMENTS |
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We thank Christine Kretz, Anne Gansmuller and Jean-Luc Vonesh for technical help, Dr Kaoru Takegawa (Japan) for sharing many unpublished results and the SpVPS34 antibody, Dr Didier Devys for continous support, Pierre Maziere (Paris), Jean-Claude Sulpice (Paris), Anna Buj-Bello and Hélène Boeuf for useful discussions. This study was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg (HUS), and by grants from the Association Française contre les Myopathies (AFM).
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NOTE ADDED IN PROOF |
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Similar results for the lipid phosphatase activity of myotubularin have independently been obtained: Taylor et al. (2000) Proc. Natl Acad. Sci. USA, 97, 8910–8915.
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FOOTNOTES |
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+ To whom correspondence should be addresssed. Tel: +33 388 65 32 44; Fax: +33 388 65 32 46; Email: mtm@titus.u-strasbg.fr
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 A. Kihara, H. Sakuraba, M. Ikeda, A. Denpoh, and Y. Igarashi Membrane Topology and Essential Amino Acid Residues of Phs1, a 3-Hydroxyacyl-CoA Dehydratase Involved in Very Long-chain Fatty Acid Elongation J. Biol. Chem., April 25, 2008; 283(17): 11199 - 11209. [Abstract] [Full Text] [PDF]
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F. L. Robinson, I. R. Niesman, K. K. Beiswenger, and J. E. Dixon Loss of the inactive myotubularin-related phosphatase Mtmr13 leads to a Charcot-Marie-Tooth 4B2-like peripheral neuropathy in mice PNAS, March 25, 2008; 105(12): 4916 - 4921. [Abstract] [Full Text] [PDF]
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V. Tosch, H. M. Rohde, H. Tronchere, E. Zanoteli, N. Monroy, C. Kretz, N. Dondaine, B. Payrastre, J.-L. Mandel, and J. Laporte A novel PtdIns3P and PtdIns(3,5)P2 phosphatase with an inactivating variant in centronuclear myopathy Hum. Mol. Genet., November 1, 2006; 15(21): 3098 - 3106. [Abstract] [Full Text] [PDF]
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N. Fili, V. Calleja, R. Woscholski, P. J. Parker, and B. Larijani Compartmental signal modulation: Endosomal phosphatidylinositol 3-phosphate controls endosome morphology and selective cargo sorting PNAS, October 17, 2006; 103(42): 15473 - 15478. [Abstract] [Full Text] [PDF]
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 O. Lorenzo, S. Urbe, and M. J. Clague Systematic analysis of myotubularins: heteromeric interactions, subcellular localisation and endosomerelated functions J. Cell Sci., July 15, 2006; 119(14): 2953 - 2959. [Abstract] [Full Text] [PDF]
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 J. E. Duex, F. Tang, and L. S. Weisman The Vac14p-Fig4p complex acts independently of Vac7p and couples PI3,5P2 synthesis and turnover J. Cell Biol., February 27, 2006; 172(5): 693 - 704. [Abstract] [Full Text] [PDF]
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M. J. Begley, G. S. Taylor, M. A. Brock, P. Ghosh, V. L. Woods, and J. E. Dixon Molecular basis for substrate recognition by MTMR2, a myotubularin family phosphoinositide phosphatase PNAS, January 24, 2006; 103(4): 927 - 932. [Abstract] [Full Text] [PDF]
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 S. Noguchi, M. Fujita, K. Murayama, R. Kurokawa, and I. Nishino Gene expression analyses in X-linked myotubular myopathy Neurology, September 13, 2005; 65(5): 732 - 737. [Abstract] [Full Text] [PDF]
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F. L. Robinson and J. E. Dixon The Phosphoinositide-3-phosphatase MTMR2 Associates with MTMR13, a Membrane-associated Pseudophosphatase Also Mutated in Type 4B Charcot-Marie-Tooth Disease J. Biol. Chem., September 9, 2005; 280(36): 31699 - 31707. [Abstract] [Full Text] [PDF]
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M. Pele, L. Tiret, J.-L. Kessler, S. Blot, and J.-J. Panthier SINE exonic insertion in the PTPLA gene leads to multiple splicing defects and segregates with the autosomal recessive centronuclear myopathy in dog Hum. Mol. Genet., July 1, 2005; 14(13): 1905 - 1906. [Full Text] [PDF]
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M. Pele, L. Tiret, J.-L. Kessler, S. Blot, and J.-J. Panthier SINE exonic insertion in the PTPLA gene leads to multiple splicing defects and segregates with the autosomal recessive centronuclear myopathy in dogs Hum. Mol. Genet., June 1, 2005; 14(11): 1417 - 1427. [Abstract] [Full Text] [PDF]
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O. Lorenzo, S. Urbe, and M. J. Clague Analysis of phosphoinositide binding domain properties within the myotubularin-related protein MTMR3 J. Cell Sci., May 1, 2005; 118(9): 2005 - 2012. [Abstract] [Full Text] [PDF]
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 J. Zhang, C.-h. Wong, W. Xia, D. D. Mruk, N. P. Y. Lee, W. M. Lee, and C. Y. Cheng Regulation of Sertoli-Germ Cell Adherens Junction Dynamics via Changes in Protein-Protein Interactions of the N-Cadherin-{beta}-Catenin Protein Complex which Are Possibly Mediated by c-Src and Myotubularin-Related Protein 2: An in Vivo Study Using an Androgen Suppression Model Endocrinology, March 1, 2005; 146(3): 1268 - 1284. [Abstract] [Full Text] [PDF]
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 N. A. Faustino and T. A. Cooper Identification of Putative New Splicing Targets for ETR-3 Using Sequences Identified by Systematic Evolution of Ligands by Exponential Enrichment Mol. Cell. Biol., February 1, 2005; 25(3): 879 - 887. [Abstract] [Full Text] [PDF]
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 D. D. Mruk and C. Y. Cheng Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis Endocr. Rev., October 1, 2004; 25(5): 747 - 806. [Abstract] [Full Text] [PDF]
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K. Tsujita, T. Itoh, T. Ijuin, A. Yamamoto, A. Shisheva, J. Laporte, and T. Takenawa Myotubularin Regulates the Function of the Late Endosome through the GRAM Domain-Phosphatidylinositol 3,5-Bisphosphate Interaction J. Biol. Chem., April 2, 2004; 279(14): 13817 - 13824. [Abstract] [Full Text] [PDF]
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H. Tronchere, J. Laporte, C. Pendaries, C. Chaussade, L. Liaubet, L. Pirola, J.-L. Mandel, and B. Payrastre Production of Phosphatidylinositol 5-Phosphate by the Phosphoinositide 3-Phosphatase Myotubularin in Mammalian Cells J. Biol. Chem., February 20, 2004; 279(8): 7304 - 7312. [Abstract] [Full Text] [PDF]
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K. Wu, M. E. Bottazzi, C. de la Fuente, L. Deng, S. D. Gitlin, A. Maddukuri, S. Dadgar, H. Li, A. Vertes, A. Pumfery, et al. Protein Profile of Tax-associated Complexes J. Biol. Chem., January 2, 2004; 279(1): 495 - 508. [Abstract] [Full Text] [PDF]
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H. Dang, Z. Li, E. Y. Skolnik, and H. Fares Disease-related Myotubularins Function in Endocytic Traffic in Caenorhabditis elegans Mol. Biol. Cell, January 1, 2004; 15(1): 189 - 196. [Abstract] [Full Text] [PDF]
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 C. Chaussade, L. Pirola, S. Bonnafous, F. Blondeau, S. Brenz-Verca, H. Tronchere, F. Portis, S. Rusconi, B. Payrastre, J. Laporte, et al. Expression of Myotubularin by an Adenoviral Vector Demonstrates Its Function as a Phosphatidylinositol 3-Phosphate [PtdIns(3)P] Phosphatase in Muscle Cell Lines: Involvement of PtdIns(3)P in Insulin-Stimulated Glucose Transport Mol. Endocrinol., December 1, 2003; 17(12): 2448 - 2460. [Abstract] [Full Text] [PDF]
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J. Laporte, F. Bedez, A. Bolino, and J.-L. Mandel Myotubularins, a large disease-associated family of cooperating catalytically active and inactive phosphoinositides phosphatases Hum. Mol. Genet., October 15, 2003; 12(90002): R285 - 292. [Abstract] [Full Text] [PDF]
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P. Berger, C. Schaffitzel, I. Berger, N. Ban, and U. Suter Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module PNAS, October 14, 2003; 100(21): 12177 - 12182. [Abstract] [Full Text] [PDF]
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S. Merlot, R. Meili, D. J. Pagliarini, T. Maehama, J. E. Dixon, and R. A. Firtel A PTEN-related 5-Phosphatidylinositol Phosphatase Localized in the Golgi J. Biol. Chem., October 10, 2003; 278(41): 39866 - 39873. [Abstract] [Full Text] [PDF]
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Y. Xue, H. Fares, B. Grant, Z. Li, A. M. Rose, S. G. Clark, and E. Y. Skolnik Genetic Analysis of the Myotubularin Family of Phosphatases in Caenorhabditis elegans J. Biol. Chem., September 5, 2003; 278(36): 34380 - 34386. [Abstract] [Full Text] [PDF]
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H. H. Nandurkar, M. Layton, J. Laporte, C. Selan, L. Corcoran, K. K. Caldwell, Y. Mochizuki, P. W. Majerus, and C. A. Mitchell Identification of myotubularin as the lipid phosphatase catalytic subunit associated with the 3-phosphatase adapter protein, 3-PAP PNAS, July 22, 2003; 100(15): 8660 - 8665. [Abstract] [Full Text] [PDF]
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S.-A Kim, P. O. Vacratsis, R. Firestein, M. L. Cleary, and J. E. Dixon Regulation of myotubularin-related (MTMR)2 phosphatidylinositol phosphatase by MTMR5, a catalytically inactive phosphatase PNAS, April 15, 2003; 100(8): 4492 - 4497. [Abstract] [Full Text] [PDF]
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A. Buj-Bello, V. Laugel, N. Messaddeq, H. Zahreddine, J. Laporte, J.-F. Pellissier, and J.-L. Mandel The lipid phosphatase myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice PNAS, November 12, 2002; 99(23): 15060 - 15065. [Abstract] [Full Text] [PDF]
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 C. Y. Cheng and D. D. Mruk Cell Junction Dynamics in the Testis: Sertoli-Germ Cell Interactions and Male Contraceptive Development Physiol Rev, October 1, 2002; 82(4): 825 - 874. [Abstract] [Full Text] [PDF]
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A. Buj-Bello, D. Furling, H. Tronchere, J. Laporte, T. Lerouge, G. S. Butler-Browne, and J.-L. Mandel Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells Hum. Mol. Genet., September 15, 2002; 11(19): 2297 - 2307. [Abstract] [Full Text] [PDF]
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P. Berger, S. Bonneick, S. Willi, M. Wymann, and U. Suter Loss of phosphatase activity in myotubularin-related protein 2 is associated with Charcot-Marie-Tooth disease type 4B1 Hum. Mol. Genet., June 15, 2002; 11(13): 1569 - 1579. [Abstract] [Full Text] [PDF]
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R. Firestein and M. L. Cleary Pseudo-phosphatase Sbf1 contains an N-terminal GEF homology domain that modulates its growth regulatory properties J. Cell Sci., March 10, 2002; 114(16): 2921 - 2927. [Abstract] [Full Text] [PDF]
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S.-A Kim, G. S. Taylor, K. M. Torgersen, and J. E. Dixon Myotubularin and MTMR2, Phosphatidylinositol 3-Phosphatases Mutated in Myotubular Myopathy and Type 4B Charcot-Marie-Tooth Disease J. Biol. Chem., February 1, 2002; 277(6): 4526 - 4531. [Abstract] [Full Text] [PDF]
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J. Laporte, F. Blondeau, A. Gansmuller, Y. Lutz, J.-L. Vonesch, and J.-L. Mandel The PtdIns3P phosphatase myotubularin is a cytoplasmic protein that also localizes to Rac1-inducible plasma membrane ruffles J. Cell Sci., January 8, 2002; 115(15): 3105 - 3117. [Abstract] [Full Text] [PDF]
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H. H. Nandurkar, K. K. Caldwell, J. C. Whisstock, M. J. Layton, E. A. Gaudet, F. A. Norris, P. W. Majerus, and C. A. Mitchell Characterization of an adapter subunit to a phosphatidylinositol (3)P 3-phosphatase: Identification of a myotubularin-related protein lacking catalytic activity PNAS, August 14, 2001; 98(17): 9499 - 9504. [Abstract] [Full Text] [PDF]
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