Characterization of the Myotubularin Dual Specificity Phosphatase Gene Family from Yeast to Human

Abstract

X-linked myotubular myopathy (XLMTM) is a severe congenital muscle disorder due to mutations in the MTM1 gene. The corresponding protein, myotubularin, contains the consensus active site of tyrosine phosphatases (PTP) but otherwise shows no homology to other phosphatases. Myotubularin is able to hydrolyze a synthetic analogue of tyrosine phosphate, in a reaction inhibited by orthovanadate, and was recently shown to act on both phosphotyrosine and phosphoserine. This gene is conserved down to yeast and strong homologies were found with human ESTs, thus defining a new dual specificity phosphatase (DSP) family. We report the presence of novel members of the MTM gene family in Schizosaccharomyces pombe, Caenorhabditis elegans, zebrafish, Drosophila, mouse and man. This represents the largest family of DSPs described to date. Eight MTM-related genes were found in the human genome and we determined the chromosomal localization and expression pattern for most of them. A subclass of the myotubularin homologues lacks a functional PTP active site. Missense mutations found in XLMTM patients affect residues conserved in a Drosophila homologue. Comparison of the various genes allowed construction of a phylogenetic tree and reveals conserved residues which may be essential for function. These genes may be good candidates for other genetic diseases.

Myotubularin, a protein tyrosine phosphatase mutated in myotubular myopathy, dephosphorylates the lipid second messenger, phosphatidylinositol 3-phosphate

“…To this end, we have expressed recombinant human myotubularin in E. coli as a fusion protein (GST-MTM1-His 6 ) with N-terminal glutathione S-transferase and C-terminal six-histidine tags for affinity purification. As previously shown, the recombinant myotubularin fusion protein is active toward the artificial PTP substrate para-nitrophenylphosphate (pNPP) (8). We have found that myotubularin hydrolyzes pNPP at a pH optimum of 6.0 with a K m of 21 mM, a k cat of 0.4 s Ϫ1 , and a k cat ͞K m of 20 s Ϫ1 ⅐M Ϫ1 .…”

“…Although previous studies have demonstrated that recombinant myotubularin possesses dual specificity protein phosphatase activity, its relative efficiency toward these substrates has not been reported (7,8). To this end, we have expressed recombinant human myotubularin in E. coli as a fusion protein (GST-MTM1-His 6 ) with N-terminal glutathione S-transferase and C-terminal six-histidine tags for affinity purification.…”

“…The myotubularin gene (MTM1), which is mutated in X-linked myotubular myopathy, encodes a protein with sequence similarity to dual specificity protein tyrosine phosphatases (5). Myotubularin contains the Cys-X 5 -Arg (CX 5 R) active site motif that is the hallmark of the protein tyrosine phosphatase (PTP) superfamily and exhibits dual specificity protein phosphatase activity in vitro (6)(7)(8)(9). In addition, myotubularin-related proteins are conserved among eukaryotes, suggesting a common substrate or function (5,9).…”

Regulation of myotubularin-related (MTMR)2 phosphatidylinositol phosphatase by MTMR5, a catalytically inactive phosphatase

“…Initially, MTM1 was reported to be a dual-specificity phosphatase (4)(5)(6). However, we and others have demonstrated that MTM1 utilizes the lipid second messenger, phosphatidylinositol 3-phosphate [PI(3)P], as a physiological substrate (7,8).…”

“…Type 4B1 Charcot-Marie-Tooth disease is an autosomal recessive demyelinating neuropathy characterized by abnormally folded myelin sheaths and Schwann cell proliferation in peripheral nerves (23,24). MTM1 and MTMR2 are highly similar proteins (64% identity, 76% similarity), use the same physiologic substrate, and have a ubiquitous expression pattern (6,(9)(10)(11)(12). However, mutations in MTM1 and MTMR2 cause different diseases with different target tissues and pathological characteristics.…”