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

Abstract

The myotubularin (MTM) family constitutes one of the most highly conserved protein-tyrosine phosphatase subfamilies in eukaryotes. MTM1, the archetypal member of this family, is mutated in X-linked myotubular myopathy, whereas mutations in the MTM-related (MTMR)2 gene cause the type 4B1 Charcot-Marie-Tooth disease, a severe hereditary motor and sensory neuropathy. In this study, we identified a protein that specifically interacts with MTMR2 but not MTM1. The interacting protein was shown by mass spectrometry to be MTMR5, a catalytically inactive member of the MTM family. We also demonstrate that MTMR2 interacts with MTMR5 via its coiled-coil domain and that mutations in the coiled-coil domain of either MTMR2 or MTMR5 abrogate this interaction. Through this interaction, MTMR5 increases the enzymatic activity of MTMR2 and dictates its subcellular localization. This article demonstrates an active MTM member being regulated by an inactive family member.

Figure 1

Identification of an MTMR2-interacting protein. (A) 293 cells were transfected with either FLAG-MTM1 or FLAG-MTMR2 expression vectors. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation by using anti-FLAG antibody. Immunoprecipitated proteins were separated by SDS/PAGE, and the gel was stained with Coomassie blue. The p200 band excised for MALDI/TOF mass spectrometry analysis is marked with an arrow. (B) MALDI/TOF mass spectrometry of the typtic digests of MTMR5. Shown in bold are peaks unique to the p200 band compared with control samples, which correspond to matched peptides to human MTMR5. The peptide masses analyzed by MALDI/post source decay are underlined. (C) 293 cells were transfected with FLAG-MTM1 or FLAG-MTMR2 expression vectors. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation by using anti-FLAG antibody, and endogenous MTMR5 was detected by Western blot analysis by using anti-MTMR5 antibody. (D) Purified GST-tagged bacterial recombinant MTMR5 (10 μg) was incubated with equimolar His-tagged MTM1 or MTMR2 fusion proteins. As a control, equimolar GST was incubated with His-tagged MTMR2. GST or GST-MTMR5 was pulled down by glutathione-agarose beads. Coprecipitated proteins were resolved on SDS/PAGE and detected by Western blot analysis by using anti-His antibody.

Figure 2

MTMR2 forms homo-/heterodimer through a coiled-coil interaction. (A) Structural features of MTM1, MTMR2, and MTMR5. MTM1 and MTMR2 contain a catalytic domain that encompasses the CX₅R active site motif of PTP. In addition, MTM proteins possess several other domains/motifs that are likely to facilitate membrane association and protein–protein interactions. MTM1 and MTMR2 contain a PH domain, a coiled-coil domain, and a PDZ-binding motif as indicated. MTMR5 also has a coiled-coil domain followed by a PH domain in its C-terminal region. Domain boundaries were obtained from smart (34) and coil (35). (B) 293 cells were transfected with the indicated expression vectors. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation by using anti-GFP antibody. Coprecipitated endogenous MTMR5 was analyzed by Western blot analysis by using anti-MTMR5 antibody (Upper). The expression level of proteins in the transfected cells was monitored by Western blot analysis (Lower). (C and D) pEGFP-MTMR2 (WT) expression vectors were cotransfected with various FLAG-tagged MTMR5 mutant expression vectors (C), or FLAG-MTMR2 (WT) expression vectors were cotransfected with various EGFP-tagged MTMR2 mutant expression vectors (D) as indicated. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation by using anti-FLAG antibody followed by Western blot analysis by using anti-GFP antibody (Top). The expression level of proteins in the transfected cells was monitored by Western blot analysis (Middle and Bottom).

Figure 3

MTMR2 point mutants diminish its homo-/heterodimerization. (A) Sequence alignment of coiled-coil domain of MTM1 and MTMR2. Gray boxes represent conserved amino acids. The heptad repeat is denoted with the letters a–g, with leucine residues residing at the d position. (B and C) 293 cells were transfected with FLAG-tagged MTMR2 point mutant expression vectors alone (B) or combined with EGFP-MTMR2 (WT) expression vectors (C) as indicated. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation by using anti-FLAG antibody followed by Western blot analysis by using anti-MTMR5 (B) or anti-GFP (C) antibody. The expression level of proteins in the transfected cells was monitored by Western blot analysis with anti-FLAG and/or anti-GFP antibody.

Figure 4

MTMR5 regulates the subcellular localization of MTMR2. COS-1 cells were cotransfected with EGFP-MTMR2 (WT or Δ-coil) in combination with FLAG-MTMR5 (WT, Δ-PH, or Δ-coil) expression vectors. Thirty hours after transfection, cells were fixed and immunostained by using anti-FLAG antibody. Green fluorescent staining for EGFP-MTMR2 (WT and Δ-coil) is shown in A, C, E, and G. Red fluorescent staining for Cy3-conjugated anti-mouse secondary antibody bound to the FLAG-MTMR5 (WT, Δ-PH, and Δ-coil) is shown in B, D, F, and H.