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

Abstract

The lipid second messenger phosphatidylinositol 3-phosphate [PI(3)P] plays a crucial role in intracellular membrane trafficking. We report here that myotubularin, a protein tyrosine phosphatase required for muscle cell differentiation, is a potent PI(3)P phosphatase. Recombinant human myotubularin specifically dephosphorylates PI(3)P in vitro. Overexpression of a catalytically inactive substrate-trapping myotubularin mutant (C375S) in human 293 cells increases PI(3)P levels relative to that of cells overexpressing the wild-type enzyme, demonstrating that PI(3)P is a substrate for myotubularin in vivo. In addition, a Saccharomyces cerevisiae strain in which the myotubularin-like gene (YJR110w) is disrupted also exhibits increased PI(3)P levels. Both the recombinant yeast enzyme and a human myotubularin-related protein (KIAA0371) are able to dephosphorylate PI(3)P in vitro, suggesting that this activity is intrinsic to all myotubularin family members. Mutations in the MTM1 gene that cause human myotubular myopathy dramatically reduce the ability of the phosphatase to dephosphorylate PI(3)P. Our findings provide evidence that myotubularin exerts its effects during myogenesis by regulating cellular levels of the inositol lipid PI(3)P.X -linked myotubular myopathy is a severe congenital disorder in which the muscle cells of affected individuals contain large, centrally placed nuclei and structural features characteristic of fetal myotubes, suggesting that differentiation has been arrested at a step preceding myofiber formation (1-4). 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 (6-9). In addition, myotubularin-related proteins are conserved among eukaryotes, suggesting a common substrate or function (5, 9). However, the physiologic target(s) of myotubularin and its essential role in myogenic development have yet to be identified.Phosphoinositides produced by the actions of phosphatidylinositol (PI) 3-kinases play key roles in a diverse array of cellular processes, including responses to extracellular agonists, growth, survival, cytoskeletal organization, differentiation, and membrane trafficking (10)(11)(12)(13)(14). Recently, the PTEN͞MMAC1 gene was identified as a candidate tumor suppressor gene, which mapped to chromosome 10q23, a region frequently mutated in a variety of tumors (15, 16). The PTEN͞MMAC1 gene encodes a protein with similarity to dual specificity protein tyrosine phosphatases (15,16). Our laboratory has shown that PTEN (phosphatase and tensin homolog) dephosphorylates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PI (3,4,5)P 3 ), thus identifying it as the first PTP superfamily enzyme that utilizes an inositol lipid as its physiologic substr...

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

“…(EGFP)-tagged MTM1 and MTMR2 fusion proteins have been described (7,11). Bacterial expression vectors for recombinant His-tagged MTM1 and MTMR2 fusion proteins have also been described (7,11).…”

“…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). Recent findings demonstrate that other MTMR phosphatases MTMR1, MTMR2, MTMR3, MTMR4, and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this substrate is common to all active MTM family members (9-12).…”

Phosphoinositides in Constitutive Membrane Traffic

“…There are eight myotubularin-related genes in humans and one in S. cerevisiae (YMR1). Recombinant MTM1, MTMR2, MTMR3 (KIAA0371), MTMR6, and Ymr1p proteins are active PtdIns(3)P phosphatases that also hydrolyze PtdIns(3,5)P 2 (309,350). The activity of MTM1 toward PtdIns(5)P in vitro is 200-fold less than for PtdIns(3)P, and other phosphoinositides are significantly poorer substrates (350).…”

Depression of voltage-activated Ca2+release in skeletal muscle by activation of a voltage-sensing phosphatase

“…The plasma membrane level of PtdIns(4,5)P 2 was also shown to be increased by hyperosmolarity in several cell types including cardiac cells (Nasuhoglu et al, 2002), which may occur in skeletal muscle during high intensity exercise (Lindinger et al, 1992). The potential for alteration in phosphoinositide metabolism to trigger specific muscle diseases was demonstrated by the fact that deficiency in the phosphatidylinositolphosphate Mtm1 is responsible for myotubular myopathy (Taylor et al, 2000). Interestingly, there is strong evidence that defective SR Ca 2+ release is primarily involved in this depolarizing pulses is likely to correlate with a substantial reduction of the membrane-bound PtdIns(4,5)P 2 level.…”