Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway

Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological featu res suggesting an impairment in maturation of muscle fibres, The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consens us. It defines a family of at least nine genes in man, including the antiph osphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive for m of Charcot-Marie-Tooth disease. Myotubularin shows a dual specificity pro tein phosphatase activity in vitro. We have performed an in vivo test of ty rosine phosphatase activity in Schizosaccharomyces pombe, indicating that m yotubularin does not have a broad specificity tyrosine phosphatase activity . Expression of active human myotubularin inhibited growth of S.pombe and i nduced a vacuolar phenotype similar to that of mutants of the vacuolar prot ein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylin ositol 3-kinase (P13K). In S.pombe cells deleted for the endogenous MTM hom ologous gene, expression of human myotubularin decreased the level of phosp hatidylinositol 3-phosphate (P13P). 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 k inase activity. Wild-type myotubularin was also able to directly dephosphor ylate P13P and P14P in vitro. Myotubularin may thus decrease P13P levels by down-regulating P13K activity and by directly degrading P13P.