Production of Phosphatidylinositol 5-Phosphate by the Phosphoinositide 3-Phosphatase Myotubularin in Mammalian Cells

MTM1, the gene encoding myotubularin (MTM1), is mutated in the X-linked myotubular myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of myotubular myopathy. MTM1, the gene encoding myotubularin (MTM1), is mutated in the X-linked myotubular myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of myotubular myopathy. MTM1 1The abbreviations used are: MTM1, myotubularin; PI, phosphoinositides; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol 3-phosphate, PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; MTMR, myotubularin-related proteins; XLMTM, X-linked genetic disorder myotubular myopathy; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GST, glutathione S-transferase; GFP, green fluorescent protein; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)); FYVE, Fab1p/YOTB/Vac1p/EEA1; HPLC, high pressure liquid chromatography. 1The abbreviations used are: MTM1, myotubularin; PI, phosphoinositides; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol 3-phosphate, PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; MTMR, myotubularin-related proteins; XLMTM, X-linked genetic disorder myotubular myopathy; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GST, glutathione S-transferase; GFP, green fluorescent protein; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)); FYVE, Fab1p/YOTB/Vac1p/EEA1; HPLC, high pressure liquid chromatography. is a 66-kDa ubiquitous protein that is found mutated in the X-linked genetic disorder myotubular myopathy (XLMTM). The disease, which affects 1 of 50,000 newborn males, is characterized by muscle weakness and hypotonia at birth. In the most severe cases, it leads to the death in the first weeks of life because of respiratory failure. More than 198 different mutations have been found in the MTM1 gene, mainly leading to the absence or the expression of a truncated version of the protein (1Biancalana V. Caron O. Gallati S. Baas F. Kress W. Novelli G. D'Apice M.R. Lagier-Tourenne C. Buj-Bello A. Romero N.B. Mandel J.-L. Hum. Genet. 2003; 112: 135-142Crossref PubMed Scopus (98) Google Scholar). The gene encoding MTM1 was cloned in 1996 (2Laporte J. Hu L.J. Kretz C. Mandel J.-L. Kioschis P. Coy J.F. Klauck S.M. Poustka A. Dahl N. Nat. Genet. 1996; 13: 175-182Crossref PubMed Scopus (502) Google Scholar), and its sequence shows the presence of the HCSDGWDRT motif, which fits the conserved consensus sequence CX5R of the dual specificity phosphatase/phosphotyrosine phosphatase (DSP/PTP) family. However, studies on MTM1 activity showed that its preferred substrate is not a tyrosine-phosphorylated protein but the phosphoinositide PtdIns(3)P (3Blondeau F. Laporte J. Bodin S. Superti-Furga G. Payrastre B. Mandel J.-L. Hum. Mol. Genet. 2000; 9: 2223-2229Crossref PubMed Scopus (207) Google Scholar, 4Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (273) Google Scholar), thereby identifying MTM1 as a lipid phosphatase. Thus, MTM1 is a 3-phosphatase but exhibits a different substrate specificity compared with the tumor suppressor PTEN that hydrolyzes preferentially PtdIns(3,4,5)P3 (5Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2552) Google Scholar). MTM1 could therefore act as a regulator of the highly active phosphoinositide (PI) metabolism in cells (6Tronchère H. Buj-Bello A. Mandel J.-L. Payrastre B. Cell. Mol. Life Sci. 2003; 60: 2084-2099Crossref PubMed Scopus (21) Google Scholar), where interconversions of the eight PIs described so far is controlled by a set of specific kinases and phosphatases (7Payrastre B. Missy K. Giuriato S. Bodin S. Plantavid M. Gratacap M. Cell. Signal. 2001; 13: 377-387Crossref PubMed Scopus (187) Google Scholar). PIs are minor phospholipids of the cell membrane; however, they play a major role of second messengers in diverse cellular functions such as proliferation, apoptosis, cytoskeletal remodeling, and vesicular trafficking (7Payrastre B. Missy K. Giuriato S. Bodin S. Plantavid M. Gratacap M. Cell. Signal. 2001; 13: 377-387Crossref PubMed Scopus (187) Google Scholar, 8Toker A. Cell. Mol. Life Sci. 2002; 59: 761-779Crossref PubMed Scopus (177) Google Scholar). The way PIs act is via targeting of proteins to specific membrane locations by binding to specific protein domains such as the pleckstrin homology, phox, Fab1p/YOTB/Vac1p/EEA1 (FYVE), epsin N-terminal homology, and band 4.1/ezrin/radixin/moesin domains. MTM1 is highly conserved during evolution and is the founding member of the myotubularin-related proteins (MTMRs) family, which today counts 14 members. In addition to MTM1, other members of the family are linked to genetic diseases. MTMR1 is a target for aberrant splicing in the congenital myotonic dystrophy, cDM1 (9Buj-Bello A. Furling D. Tronchère H. Laporte J. Lerouge T. Buttler-Browne G.S. Mandel J.-L. Hum. Mol. Genet. 2002; 11: 2297-2307Crossref PubMed Scopus (107) Google Scholar), and mutations in MTMR2 have been associated to the Charcot-Marie-Tooth disease type 4B1 (CMT4B1), a demyelinating neuropathy (10Bolino 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 (414) Google Scholar). Two members of the phosphatase-dead MTMR subgroup have also been implicated in defects or disease, respectively. MTMR5/SBF1 was shown to play a role in spermatogenesis in mice (11Firestein R. Nagy P.L. Daly M. Huie P. Conti M. Cleary M.L. J. Clin. Investig. 2002; 109: 1165-1172Crossref PubMed Scopus (63) Google Scholar), and MTMR13/SBF2 was recently found mutated in the CMT4B2, another type of Charcot-Marie-Tooth disease (12Senderek J. Bergmann C. Weber S. Ketelsen U.-P. Schorle H. Rudnik-Schöneborn S. Buttner R. Buchheim E. Zerres K. Hum. Mol. Genet. 2003; 12: 349-356Crossref PubMed Scopus (219) Google Scholar, 13Azzedine H. Bolino A. Taïeb T. Birouk N. Di Duca M. Bouhouche A. Benamou S. Mrabet A. Hammadouche T. Chkili T. Gouider R. Ravazzolo R. Brice A. Laporte J. LeGuern E. Am. J. Hum. Genet. 2003; 72: 1141-1153Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Thus, although the cellular function of the MTM family members appears essential, it still remains elusive. Recent studies (14Buj-Bello A. Laugel V. Messaddeq N. Zahreddine H. Laporte J. Pellissier J.F. Mandel J.-L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15060-15065Crossref PubMed Scopus (164) Google Scholar) on the knock-out MTM1 mice proposed a role for the phosphatase in the maintenance of muscle fiber state rather than in myogenesis to explain the physiopathological mechanism of the XLMTM. As a 3-phosphatase hydrolyzing PtdIns(3)P, MTM1 was proposed to play a regulatory role in vesicular trafficking, and in some cases, overexpression of the phosphatase indeed leads to the disappearance of PtdIns(3)P endosomal punctate labeling (15Kim S.-A. Taylor G.S. Torgersen K.M. Dixon J.E. J. Biol. Chem. 2002; 277: 4526-4531Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). However, more studies are necessary to MTM1 to a role in vesicular this we whether endogenous MTM1 could affect the of PtdIns(3)P in early endosomes by established cell lines from patients from myotubular myopathy. results that in the XLMTM cell a for early and PtdIns(3)P labeling are not significantly affected compared with cell we MTM1 cellular function and specificity other been shown recently that members of the MTM family, in the yeast and the MTMR2 in an in vitro assay, can as a substrate in addition to PtdIns(3)P, leading to the production of PtdIns(5)P D.M. S. D. G. Biol. 2001; 11: Full Text Full Text PDF PubMed Scopus Google Scholar, P. S. S. M. U. Hum. Mol. Genet. 2002; 11: PubMed Scopus Google Scholar), a described PubMed Scopus Google Scholar). we that both MTM1 and MTMR1 are also to hydrolyze in in addition to we demonstrate that MTM1 is a of PtdIns(5)P in vitro and in to a role for the phosphatase in the production of this recently described in mammalian cells. data that MTM1 could affect a minor of PtdIns(3)P and that the production of PtdIns(5)P, its as a might be to its cellular and was by of MTM1 and into a with an N-terminal the other described (9Buj-Bello A. Furling D. Tronchère H. Laporte J. Lerouge T. Buttler-Browne G.S. Mandel J.-L. Hum. Mol. Genet. 2002; 11: 2297-2307Crossref PubMed Scopus (107) Google Scholar, J. F. Tronchère H. Mandel Payrastre B. 2002; PubMed Scopus Google Scholar). The was an N-terminal of MTM1 J. F. A. Mandel J.-L. J. Sci. 2002; PubMed Google Scholar), and the was from The for the with PtdIns(3)P was a of H. for M. R. H. J. 2000; PubMed Scopus Google Scholar). The protein was in to a and in with reduced The protein was the the and the was with type was by N. The The type was in the E. and on the type or the substrate generated by as described in C. S. F. S. Tronchère H. F. S. Payrastre B. Laporte J. E. Mol. 2003; PubMed Scopus Google Scholar). and and L6 cells at with in Dulbecco's modified Eagle's and L6 to myotubes was induced by cells for in and in PBS, at and of was of the L6 myotubes at a of of of in the presence of the was and cells for shock was by the cells in for at or cells in and with of to the cells in and used for cells in and into of 1 of cells cells in and used for PtdIns(5)P mass cell lines from XLMTM patients described J. Kress W. Mandel J.-L. 2001; PubMed Scopus Google Scholar) and by O. W. Kress H. O. E. and N. In in with or protein for at The was in and in and for phosphatase activity in with or fluorescent to Taylor and Dixon G.S. Dixon J.E. 2001; PubMed Scopus Google Scholar). to and J. 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J. 2000; PubMed Scopus Google Scholar). we the role of MTM1 in the regulation of the PtdIns(3)P by its in and XLMTM patients cell In the control cell is a of PtdIns(3)P by the with of its the early by a specific PtdIns(3)P labeling was in the cell for the MTM1 gene, as as in cell lines and established from patients affected during by a myopathy XLMTM but with mutations in the MTM1 gene and by a respectively. are related with a and an In the cell lines of MTM1 and and in the cell J. Kress W. Mandel J.-L. 2001; PubMed Scopus Google Scholar), is of PtdIns(3)P labeling and which still with the and also PtdIns(3)P in established cell lines from XLMTM patients a cell more to the severe muscular in XLMTM In cell a in MTM1 level compared with J. Kress W. Mandel J.-L. 2001; PubMed Scopus Google Scholar), and of from patients severe and been by the Our results the of PtdIns(3)P with the in the cell lines from XLMTM patients and and the control As a shows the of PtdIns(3)P by in data that the lack of expression of endogenous MTM1 in or not significantly affect the of PtdIns(3)P on and PtdIns(3)P in and XLMTM and from patients on and for studies as described The cell lines used are as XLMTM PtdIns(3)P is with the and and endogenous with an and In the and the of the with the early muscle cells with for In the and the of the with the early MTM1 a PtdIns(3)P and in from XLMTM cell lines that endogenous MTM1 act on a of because the in PtdIns(3)P upon MTM1 overexpression more than Payrastre and H. in the different cell lines that we have and is in with the results by Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (273) Google Scholar) and other functions for As shown recently that MTMR2 and the of can also hydrolyze PtdIns(3,5)P2, we this for with the in vitro of MTM1 phosphatase lipid substrate specificity fluorescent and MTM1 its in mammalian cells. In MTMR2 and used as shows that MTM family members can as a in addition to of the other PIs are for the MTM1 and MTMR1 as as MTMR2 and to hydrolyze and PtdIns(3)P, leading to the of the of at In to that the 3-phosphatase activity of MTM1 in vitro is we used a MTM1 where the in the phosphatase been mutated into shows that the of PtdIns(3)P and is specific for MTM1 phosphatase the in function of fluorescent lipid in the in vitro phosphatase MTM1 and that the phosphatase at 1 of fluorescent PtdIns(3)P or in at for both MTM1 and MTMR2 Because PIs in cell are the of PIs in might be of this we in vitro The results in that the specificity of MTM1 is of the by a green to the of during MTM1 assay, we found that both and in a MTM1 PtdIns(5)P in whether of by MTM1 and family members of the of the PtdIns(5)P type which PtdIns(5)P on of the to A. 2000; PubMed Scopus Google Scholar). the specific substrate of PtdIns(5)P type PtdIns(5)P was by other PtdIns(3)P and the specificity of the for The in this was as by not MTM1, and from cells where first for 3-phosphatase activity by The generated from and used as substrate for the PtdIns(5)P type in the presence of shows that the product generated by MTM1 and from is indeed PtdIns(5)P, as by the of in the results demonstrate that MTM1 in mammalian cells can into PtdIns(5)P in vitro. MTM1 PtdIns(5)P in whether MTM1 could increase PtdIns(5)P mass in in mammalian cells. cells used because they have a high of with MTM1 or the for this cell In the of as by of cells not of MTM1 by on cell is shown in and the of PIs and to the PtdIns(5)P type in vitro to As a cells by a phosphatase to PtdIns(5)P K. Giuriato S. T. F. J. C. Payrastre B. J. 2002; PubMed Scopus Google Scholar). As shown in MTM1 overexpression an increase of cells. of the shows the presence of as PIs a more the mass of PtdIns(5)P was by the and the to a of that MTM1 is to increase the PtdIns(5)P level in cells from a level of to cells In with the in vitro 3-phosphatase activity of MTM1, data that the can PtdIns(5)P in mammalian cells. PtdIns(5)P upon shown to be by shock in yeast and in mammalian cells. therefore the of shock on PtdIns(5)P production by MTM1 in the L6 to to myotubes and MTM1, or the inactive substrate MTM1 an leading to of C. S. F. S. Tronchère H. F. S. Payrastre B. Laporte J. E. Mol. 2003; PubMed Scopus Google Scholar). of PtdIns(5)P the in vitro of and a that expression of the inactive MTM1 mutant in cells by 50% the level of PtdIns(5)P compared with the expression of type MTM1 that MTM1 activity is In the of by labeling of the of PIs with that level is more important in the cells overexpressing the MTM1 mutant compared with type in the MTM1 gene are associated with the X-linked myotubular a severe genetic muscle disorder. The of the mutations leads to an absence or a protein However, the the the in vitro activity G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (273) Google Scholar) but not affect the protein level in cells J. Kress W. Mandel J.-L. 2001; PubMed Scopus Google Scholar), that the 3-phosphatase activity is for the role of In mammalian cells, PtdIns(3)P from the of a the of the yeast and its level is compared with the The most function of PtdIns(3)P is the regulation of trafficking binding to or early endosomal such as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) or H. H. W. Nat. Biol. 2001; PubMed Scopus Google Scholar). the of PtdIns(3)P in and from the established cell lines of XLMTM where expression of MTM1 is cells J. Kress W. Mandel J.-L. 2001; PubMed Scopus Google Scholar). of the to PtdIns(3)P demonstrated a with the early endosomal of MTM1 suggesting that the function of endogenous MTM1 is not to vesicular been that MTM1 can the endosomal of the PtdIns(3)P (15Kim S.-A. Taylor G.S. Torgersen K.M. Dixon J.E. J. Biol. Chem. 2002; 277: 4526-4531Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, C. S. F. S. Tronchère H. F. S. Payrastre B. Laporte J. E. Mol. 2003; PubMed Scopus Google Scholar). However, it be that this results from the of phosphatase that could in still the of the role of MTM1 as a could explain the lack of of the endosomal of the PtdIns(3)P by endogenous MTM1, which a high specific activity in is to be in by with could its activity or its by the phosphatase from the PtdIns(3)P it been shown recently that MTM family members could MTM1 can with which affects its M. Laporte J. J. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google can with MTMR2 but not MTM1 R. Cleary M.L. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google and can to Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). it is that MTM1 affects a minor of PtdIns(3)P in because in cells overexpressing MTM1, the level of PtdIns(3)P affected by the phosphatase is of PtdIns(3)P 4Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (273) Google Scholar, C. S. F. 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Biol. 2001; 25: PubMed Scopus Google Scholar). we described recently K. Giuriato S. T. F. J. C. Payrastre B. J. 2002; PubMed Scopus Google Scholar) the production of PtdIns(5)P by the a during of the cell by the PtdIns(5)P was also in the and shown to to protein domains and therefore to O. P. D. J. H. J. B. J. Payrastre B. G. N. J. Cell. 2003; Full Text Full Text PDF PubMed Scopus (414) Google Scholar). a specific mass A. 2000; PubMed Scopus Google Scholar), we showed that in mammalian cells, MTM1 could for the production of in cells, overexpression of MTM1 the cellular mass of upon we a in PtdIns(5)P in L6 cells that are the MTM1 mutant compared with type inactive mutant is as a substrate and we that it have the to with the endogenous MTM1 for we a 50% of PtdIns(5)P upon mutant expression in L6 cells. the in labeling of the is on the activity of the phosphatase because it is in the presence of MTM1 and not with the inactive mutant D278A. Thus, data that the 3-phosphatase MTM1 could also for the production of PtdIns(5)P in of PtdIns(3,5)P2, and that this PIs could some of its cellular As PtdIns(5)P is a recently discovered PI, and its function is still J. B. O. Biol. 2003; 13: Full Text Full Text PDF PubMed Scopus Google Scholar) have proposed that PtdIns(5)P is a specific of MTM1 activity by of MTM1 in vitro. more studies are necessary to the of the of PtdIns(3)P, PtdIns(3,5)P2, and PtdIns(5)P in the etiology of the genetic H. for the and N. for the