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.

Figure 1

PI(3)P phosphatase activity is not a general property of protein tyrosine phosphatases. The activities of tyrosine-specific (GST-p61/62n), dual-specific (VHR), and inositol lipid phosphatases (GST-PTEN) were compared with that of myotubularin (GST-MTM1-His₆). (a) Phosphatase activity of each enzyme using pNPP as substrate. (b) Phosphatase activity toward PI(3)P as substrate. Phosphatase assays were carried out at 30°C as described in Materials and Methods. Enzyme specific activity is expressed as moles of phosphate released per minute per mole of enzyme (mean ± SEM).

Figure 2

HPLC analysis of deacylated phosphoinositides from HEK 293 cells and budding yeast. Radiolabeled lipids from HEK293 cells overexpressing wild-type or mutant (C375S) myotubularin, and wild-type (GYC121) or myotubularin homolog null mutant (ΔYJR110w) budding yeast strains, were deacylated and separated by anion exchange chromatography as described in Materials and Methods. (a) Elution profile of radiolabeled gPIPs from 293 cells overexpressing wild-type (WT) myotubularin. (b) Elution profile of radiolabeled gPIPs from 293 cells overexpressing a catalytically inactive myotubularin mutant (C375S). (c) Elution profile of radiolabeled gPIPs from wild-type yeast. (d) Elution profile of radiolabeled gPIPs from ΔYJR110w yeast. The elution positions of gPI(3)P, gPI(4)P, and gPI(4,5)P₂ reference standards are indicated by arrows. Sample loadings represent equivalent total cellular protein and total radiolabeled phosphoinositides. Chromatographs are representative of four independent experiments.

Figure 3

MTM1 mutations associated with severe myotubular myopathy dramatically decrease the phosphatase activity of myotubularin. Recombinant wild-type or mutant (P205L, R241L, S376N, G378R, Y397C) myotubularin fusion proteins were tested for their ability to dephosphorylate pNPP and PI(3)P substrates. Phosphatase assays were conducted at 30°C as described in Materials and Methods. Values are expressed as percent wild-type myotubularin activity (mean ± SEM).

Figure 4

Myotubularin is localized to the cytoplasm in HEK293 cells. The subcellular distribution of transiently expressed EGFP (a) and EGFP-myotubularin fusion protein (EGFP-MTM1) (b) was determined by using immunofluorescence microscopy. Nuclei were stained with DAPI (c and d). An identical pattern of cytoplasmic staining for EGFP and EGFP-MTM1 or with FLAG-tagged myotubularin was observed in both HeLa cells and murine C2C12 myoblasts (not shown).

Figure 5

Myotubularin-like proteins contain C-terminal FYVE domains. The structural features of myotubularin and myotubularin-related proteins from human, D. melanogaster, and C. elegans are illustrated. N-terminal lipid phosphatase catalytic domains are represented as gray shaded ovals, and C-terminal FYVE domains are shown as shaded black circles.