(MTMR)2 Phosphatidylinositol Phosphatase by MTMR5, a Catalytically Inactive Phosphatase

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

Soo-A Kim*, Panayiotis O. Vacratsis*, Ron Firestein†, Michael L. Cleary†, and Jack E. Dixon*‡

*Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606; and †Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Jack E. Dixon, February 21, 2003 The myotubularin (MTM) family constitutes one of the most highly have been shown recently to cause the neurodegenerative dis- conserved protein-tyrosine phosphatase subfamilies in eu- order, type 4B1 Charcot–Marie-Tooth disease (21, 22). Type karyotes. MTM1, the archetypal member of this family, is mutated 4B1 Charcot–Marie-Tooth disease is an autosomal recessive in X-linked myotubular myopathy, whereas mutations in the MTM- demyelinating neuropathy characterized by abnormally folded related (MTMR)2 gene cause the type 4B1 Charcot–Marie-Tooth myelin sheaths and Schwann cell proliferation in peripheral disease, a severe hereditary motor and sensory neuropathy. In this nerves (23, 24). study, we identified a protein that specifically interacts with MTM1 and MTMR2 are highly similar proteins (64% identity, MTMR2 but not MTM1. The interacting protein was shown by mass 76% similarity), use the same physiologic substrate, and have a spectrometry to be MTMR5, a catalytically inactive member of the ubiquitous expression pattern (6, 9–12). However, mutations in MTM family. We also demonstrate that MTMR2 interacts with MTM1 and MTMR2 cause different diseases with different MTMR5 via its coiled-coil domain and that mutations in the coiled- target tissues and pathological characteristics. Therefore, MTM1 coil domain of either MTMR2 or MTMR5 abrogate this interaction. and MTMR2 may be subjected to differential regulatory mech- Through this interaction, MTMR5 increases the enzymatic activity anisms that preclude functional redundancy. In a previous study, of MTMR2 and dictates its subcellular localization. This article we showed that developmental expression and subcellular localization of MTM1 and MTMR2 are differentially regulated, demonstrates an active MTM member being regulated by an resulting in their utilization of specific cellular pools of PI(3)P inactive family member. (11). However, the physiologic function of MTM1 and related proteins in cell development and signaling processes remains he myotubularin (MTM) family constitutes one of the largest unknown. Therefore, studies directed toward clarifying the Tand most highly conserved protein-tyrosine phosphatase regulation of MTMR enzymes, as well as identifying downstream (PTP) subfamilies in eukaryotes (1–3). The human MTM family effectors, will be of significant value. of phosphatases includes MTM1͞MTM-related (MTMR)1͞ One of the most notable characteristics of the human MTM MTMR2, MTMR3͞MTMR4, and MTMR6͞MTMR7͞MTMR8 family is the existence of at least five catalytically inactive forms ͞ ͞ ͞ ͞ subgroups (1, 3). The consensus CX5R active site motif is found [MTMR5 (sbf1) MTMR9 (LIP-STYX) MTMR10 MTMR11 in the MTM family and the sequence “CSDGWDR” is invariant MTMR12 (3-PAP)], which contain germline substitutions in within all of the enzymatically active members of this family. catalytically essential residues within the PTP active site motif. Most PTPs use phosphoproteins as substrates and specifically Although the exact role of MTMR inactive forms is still unclear, dephosphorylate substrates containing only phosphotyrosine previous reports suggest that the ‘‘dead’’ phosphatases may sites. Other phosphatases, collectively known as dual-specificity function either as interaction modules or as naturally occurring phosphatases, are capable of removing phosphoserines͞ substrate-trapping mutants (25–27). The most extensively char- threonines and phosphotyrosines from protein substrates. acterized catalytically inactive MTMR protein is MTMR5. Al- Initially, MTM1 was reported to be a dual-specificity phos- though the cellular function of MTMR5 is unknown, Cleary and colleagues (28) have shown recently that male mice deficient for phatase (4–6). However, we and others have demonstrated that MTMR5 are infertile and azoospermic, suggesting a role MTM1 utilizes the lipid second messenger, phosphatidylinositol for MTM family proteins in spermatogenesis and germ cell 3-phosphate [PI(3)P], as a physiological substrate (7, 8). Recent differentiation. findings demonstrate that other MTMR phosphatases MTMR1, In this study, we have searched for proteins that interact with MTMR2, MTMR3, MTMR4, and MTMR6 also dephosphory- MTM1 and͞or MTMR2 by coimmunoprecipitation. The coim- late PI(3)P, suggesting that activity toward this substrate is munoprecipitated proteins were then subjected to proteolytic common to all active MTM family members (9–12). MTMR2 digestion and analyzed by mass spectrometry. By using this and MTMR3 have also been shown to dephosphorylate phos- approach, we have found that MTMR2, but not MTM1, directly phatidylinositol 3,5-bisphosphate (9, 13). PI(3)P plays a key role interacts with MTMR5. Through this interaction, MTMR5 in membrane trafficking͞vesicular transport processes and regulates the enzymatic activity and the subcellular localization serves as a targeting mechanism for proteins containing specific of MTMR2. This study demonstrates a previously unreported PI(3)P-binding modules such as Fab1͞YOTB͞Vac1p͞EEA1 functional role for catalytically inactive MTM family members. (FYVE), pleckstrin homology (PH), and Phox homology domains (14–17). Materials and Methods To date, two MTMR proteins have been associated with Plasmids. Vectors for mammalian expression of N-terminally human diseases. The MTM1 gene on chromosome Xq28 is FLAG-tagged MTM1 and MTMR2 and enhanced GFP mutated in X-linked myotubular myopathy, a severe congenital muscular disorder characterized by hypotonia and generalized muscle weakness in newborn males (18, 19). Myogenesis in Abbreviations: MTM, myotubularin; PTP, protein-tyrosine phosphatase; MTMR, MTM- ͞ related; PI(3)P, phosphatidylinositol 3-phosphate; PH, pleckstrin homology; EGFP, en- affected individuals is arrested at a late stage of differentiation hanced GFP; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight. maturation following myotube formation, and the muscle cells ‡To whom correspondence should be sent at the present address: Department of Pharma- have characteristically large centrally located nuclei (20). Mu- cology, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0636. tations in a second MTM gene MTMR2, on chromosome 11q22, E-mail: [email protected].

4492–4497 ͉ PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0431052100 Downloaded by guest on September 28, 2021 (EGFP)-tagged MTM1 and MTMR2 fusion proteins have been protein with N-terminal GST in E. coli BL21 (DE3) Codon Plus described (7, 11). Bacterial expression vectors for recombinant cells and purified by using glutathione-agarose affinity resin as His-tagged MTM1 and MTMR2 fusion proteins have also been described (33). described (7, 11). A PCR product encoding protein fragments that correspond to the MTMR2-⌬PH domain (MTMR2-⌬PH, GST Pull-Down Assay. GST-MTMR5 (10 ␮g) was added to amino acids 183–643) and the MTMR2-⌬coiled-coil domain equimolar His-tagged MTM1 and MTMR2 in reaction buffer (MTMR2-⌬coil, amino acids 1–588) were inserted into the 5Ј (20 mM Tris-Cl, pH 8.0͞200 mM NaCl͞0.1% Nonidet P-40͞1 BglII and 3Ј KpnI sites of pEGFP (CLONTECH). Site-directed mM DTT͞1 mM PMSF͞1% BSA). After a 2-h incubation at 4°C, mutagenesis was carried out by using a PCR-based procedure glutathione-agarose beads were added and incubated for an- (29). Vectors for the expression of bacterial recombinant GST- other1hat4°C. Beads were washed four times with TBST (20 tagged MTMR5 was created by using the pGEX-6P-2 (Amer- mM Tris-Cl, pH 8.0͞200 mM NaCl͞0.1% Tween 20) and boiled sham Pharmacia). The complete MTMR5 ORF was amplified by in SDS͞PAGE sample buffer. Proteins were resolved by SDS͞ PCR by using a pMSCVneo-sbf1 as a template (30). The PAGE and immunoblotted with anti-His antibody (Qiagen, pGEX-MTMR5 vector was created by inserting the MTMR5 Valencia, CA). ORF into the 5Ј-EcoRI and 3Ј-HindIII sites. Mammalian expression vectors for N-terminally FLAG-tagged full-length Immunofluorescence Microscopy. COS-1 cells were seeded on (MTMR5-WT, amino acids 1–1,930), ⌬-coiled coil (MTMR5- two-chamber slides (21 ϫ 21 mm) at a density of 5 ϫ 104 cells ⌬coil, amino acids 1–1,640), and ⌬-PH (MTMR5-⌬PH, amino per well. Cells were cultured overnight and transiently trans- acids 1–1,760) MTMR5 proteins were created by inserting cDNA fected by using FuGENE 6 reagent (Roche Molecular Bio- fragments into the 5Ј HindIII and 3Ј EcoRI sites of pCDNA3.1- chemicals). Thirty hours after transfection, the cells were NF. All constructs were confirmed by DNA sequencing. washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 10 min. After being washed with PBS for two times, Cell Culture and Immunoprecipitation. COS-1 and 293 cells were cells were incubated with methanol for 2 min. For immuno- maintained at 37°C with 5% CO2 in DMEM containing 10% staining, cells were pretreated for 30 min with 5% BSA FCS, 50 units͞ml penicillin, and 50 ␮g͞ml streptomycin. For containing PBS as a blocking agent. Then, cells were incu- immunoprecipitation, 293 cells were transiently transfected by bated with 2.5% BSA in PBS containing anti-FLAG antibody using the FuGENE 6 reagent (Roche Molecular Biochemicals) (Sigma) for 1 h and washed with PBS for 10 min. Cells were according to manufacturer protocol. Thirty hours after trans- incubated for 30 min with Cy3-conjugated anti-mouse second- fection, the cells were washed twice with PBS and lysed in RIPA ary antibody (Jackson ImmunoResearch) in 2.5% BSA con- buffer (PBS supplemented with 1% Nonidet P-40͞0.5% sodium taining PBS. Finally, the cells were washed twice for 10 min deoxycholate͞1 mM PMSF͞1 ␮g/ml aprotinin͞1 mM sodium with PBS and then mounted by using ProLong Antifade orthovanadate). The cell lysates were harvested and incubated at mounting medium (Molecular Probes). Fluorescence analysis 4°C for 30 min and cleared by centrifugation at 10,000 ϫ g for was performed by conventional fluorescence microscopy and 10 min. The supernatant was incubated with anti-FLAG anti- by confocal microscopy (Zeiss LSM 510). body (Sigma) or anti-GFP antibody (Santa Cruz Biotechnology) for 3 h, after which protein G-Sepharose (Amersham Pharma- Results and Discussion cia) was added and incubated another 1 h. The immunoprecipi- Identification of an MTMR2-Interacting Protein. To isolate and tates were washed four times with RIPA buffer containing identify proteins that interact with MTM1 and͞or MTMR2, we 0.05% SDS and boiled in SDS͞PAGE sample buffer. Proteins transfected 293 cells with either FLAG-tagged MTM1 or were resolved by SDS͞PAGE and immunoblotted with anti- MTMR2 mammalian expression vectors. FLAG-tagged fusion GFP antibody, anti-FLAG antibody, or anti-MTMR5 (Sbf1) proteins were immunoprecipitated and analyzed by SDS͞ antibody (30). PAGE and Coomassie blue staining. We observed a protein of Ϸ200 kDa (p200) that associated with MTMR2 but was absent Protein Identification by Matrix-Assisted Laser Desorption Ionization in the vector control and MTM1 samples (Fig. 1A, upper right). BIOCHEMISTRY (MALDI) Mass Spectrometry. The gel-separated protein of interest To identify this protein, MALDI͞TOF mass spectrometry was was excised and in-gel digested with trypsin as described (31). performed as described in Materials and Methods. Samples In-gel digestion was also performed on gel pieces excised from corresponding to vector control, MTM1, and MTMR2 gel a similar gel mobility region from both the vector control and lanes were individually analyzed by MALDI͞TOF. The p200 MTM1 sample. The pools of tryptic peptides from the various mass fingerprint is shown in Fig. 1B. Peptide mass values samples were analyzed by MALDI͞time-of-flight (TOF) mass unique to the p200 protein were used to search the National spectrometry in linear positive mode to generate a peptide mass Center for Biotechnology Information nonredundant data- map by using ␣-cyano-4-hydroxycinnaminic acid (saturated so- base by using either MS-FIT or PROFOUND software tools. lution in 50% acetonitrile with 0.1% trifluoroacetic acid) as the Also, MALDI͞post source decay was used to obtain partial UV-absorbing matrix. MALDI͞TOF was performed on a sequence information on two prominent peptides detected in Voyager-DE STR TOF instrument (Applied Biosystems) the MALDI͞TOF spectrum. Database searching combined equipped with a nitrogen laser operating at 337 nm. The peptide with the MALDI͞post source decay data identified MTMR5 mass values were used to search a nonredundant database (215 kDa) as the 200-kDa protein interacting specifically with (National Center for Biotechnology Information) by using MTMR2. the software tools MS-FIT (http:͞͞prospector.ucsf.edu͞) and To confirm the interaction between MTMR2 and MTMR5, PROFOUND (http:͞͞prowl.rockefeller.edu͞cgi-bin͞ProFound). 293 cells were transfected with expression vectors for FLAG- MALDI͞post source decay was performed on selected parent tagged MTM1 or MTMR2. Cells were harvested, and the cell ions in reflectron mode. extracts were subjected to immunoprecipitation by using anti- FLAG antibody, followed by Western blot analysis by using Protein Expression and Purification. Recombinant MTM1 and anti-MTMR5 antibody to detect endogenous MTMR5. As MTMR2 were expressed as C-terminally His-tagged fusion shown in Fig. 1C, MTMR5 was efficiently immunoprecipitated proteins in Escherichia coli BL21 (DE3) Codon Plus cells in the presence of MTMR2. No corresponding protein was (Stratagene) and purified by using Ni2ϩ-agarose affinity resin as observed in the vector control or MTM1 samples, confirming described (32). Recombinant MTMR5 was expressed as a fusion that the interaction was specific for MTMR2. To examine

Kim et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4493 Downloaded by guest on September 28, 2021 MTMR5 associated with WT MTMR2 and the MTMR2-PDZ mutant but failed to associate with MTMR2-⌬coil. Protein levels were verified by Western blot analysis (Fig. 2B Lower). Inversely, 293 cells were transfected with expression vectors for EGFP- tagged MTMR2 alone, together with FLAG-tagged WT MTMR5, or deletion mutants of MTMR5 as indicated in Fig. 2C. FLAG-MTMR5 proteins were immunoprecipitated by using anti-FLAG antibody and the amount of associated MTMR2 determined by Western blot analysis by using anti-GFP antibody. The MTMR5 C-terminal PH domain deletion mutant (MTMR5- ⌬PH) associated with MTMR2 at levels comparable to that of WT MTMR5. However, MTMR5-⌬coil did not associate with MTMR2, suggesting that MTMR2 and MTMR5 interact via their coiled-coil domains. To further characterize the protein–protein interactions mediated by the coiled-coil domain of MTMR2, ethylene glycol bis-(succinimidylsuccinate) cross-linking experiments were carried out by using bacterial recombinant MTM1 and MTMR2. The cross-linking experiments clearly demonstrate that MTMR2 forms a homodimer whereas MTM1 exists predominantly as a monomer (data not shown). EGFP- or FLAG-tagged MTMR2 variants were also analyzed by coimmunoprecipitation to determine the region important for Fig. 1. Identiﬁcation of an MTMR2-interacting protein. (A) 293 cells were homodimerization. Overexpressed MTMR2 was immunopre- transfected with either FLAG-MTM1 or FLAG-MTMR2 expression vectors. cipitated by using anti-FLAG antibody from each cell lysate, Thirty hours after transfection, cell extracts were subjected to immunopre- and the associated proteins were analyzed by Western blot cipitation by using anti-FLAG antibody. Immunoprecipitated proteins were analysis with anti-GFP antibody. Consistent with the in vitro separated by SDS͞PAGE, and the gel was stained with Coomassie blue. The ethylene glycol bis-(succinimidylsuccinate) cross-linking study, p200 band excised for MALDI͞TOF mass spectrometry analysis is marked with MTMR2 formed a homodimer (Fig. 2D Top). Furthermore, ͞ an arrow. (B) MALDI TOF mass spectrometry of the typtic digests of MTMR5. MTMR2 did not form a heterodimer with MTM1 (Fig. 2D Shown in bold are peaks unique to the p200 band compared with control Top). Analogous to the interaction with MTMR5, homodimer- samples, which correspond to matched peptides to human MTMR5. The ⌬ ͞ ization was impaired with MTMR2- coil (Fig. 2D Top). peptide masses analyzed by MALDI post source decay are underlined. (C) 293 ⌬ cells were transfected with FLAG-MTM1 or FLAG-MTMR2 expression vectors. Furthermore, MTMR2-PDZ mutant or MTMR2- PH did not Thirty hours after transfection, cell extracts were subjected to immunopre- affect MTMR2 dimerization, suggesting homodimerization is cipitation by using anti-FLAG antibody, and endogenous MTMR5 was de- mediated by the coiled-coil domain. tected by Western blot analysis by using anti-MTMR5 antibody. (D) Puriﬁed GST-tagged bacterial recombinant MTMR5 (10 ␮g) was incubated with The Leucine Heptad Repeat Is Critical for MTMR2 Coiled-Coil Function. equimolar His-tagged MTM1 or MTMR2 fusion proteins. As a control, equimo- Although MTM1 and MTMR2 both have putative coiled-coil lar GST was incubated with His-tagged MTMR2. GST or GST-MTMR5 was domains and share high sequence similarities, only MTMR2 pulled down by glutathione-agarose beads. Coprecipitated proteins were resolved on SDS͞PAGE and detected by Western blot analysis by using anti-His interacts with MTMR5 and forms a coiled-coil domain- antibody. dependent heterodimer. Coiled-coil domains often contain leucine residues or related amino acids spaced every seventh residue. In Fig. 3A, this heptad repeat is denoted with the letters whether the interaction between these two proteins is direct, we a–g with leucine residues residing at the d position. The MTM1 examined the binding in vitro. For the GST pull-down assay, and MTMR2 coiled-coil domains possess this heptad repeat with GST-tagged MTMR5 and His-tagged MTM1 and MTMR2 some notable differences. Specifically, the second d-position fusion proteins were expressed in bacteria and purified as leucine (L607) and several residues (K598, K603, and A605) described in Materials and Methods. As shown in Fig. 1D, found in MTMR2 are not conserved in MTM1 (Fig. 3A). MTMR2 specifically interacted with MTMR5, whereas no in- Therefore, we created MTMR2 point mutants containing these teraction was detected with MTM1. These data demonstrate that MTM1 amino acid substitutions and analyzed the ability of these MTMR2 and MTMR5 interact both in vivo and in vitro. mutants to interact with MTMR5. As shown in Fig. 3B Upper, MTMR2 K603L showed a similar binding affinity to MTMR5 compared with WT MTMR2. The binding affinity of MTMR2 MTMR2 Forms Homo-͞Heterodimer Through Its Coiled-Coil Domain. In K598M and A605D mutants was diminished. However, the addition to a phosphatase catalytic domain, MTMR proteins L607Y mutant dramatically impaired the ability of MTMR2 to possess several motifs known to mediate protein–protein and͞or associate with MTMR5. protein–lipid interactions (Fig. 2A). MTM1 and MTMR2 have When analyzed for their ability to homodimerize, the MTMR2 coiled-coil domains and PDZ-binding motifs in their C-terminal K603L and A605D mutants did not show any differences com- region that may be involved in protein–protein interactions. pared with WT MTMR2. However, the K598M and L607Y MTMR5 also has a coiled-coil domain followed by a PH domain mutants were unable to homodimerize (Fig. 3C Top). The in its C-terminal region (Fig. 2A). To identify the regions expression level of these mutants and the WT MTMR2 were ͞ mediating the MTMR2 MTMR5 interaction, EGFP-tagged equivalent (Fig. 3B Lower and 3C Middle and Bottom). These ⌬ MTMR2 with its coiled-coil domain deleted (MTMR2- coil) data show that the second leucine repeat residue, L607 of and a PDZ-binding motif mutant (MTMR2-PDZ mutant, MTMR2, is most critical for both MTMR2 homodimerization TVV3AVA) were transiently transfected into 293 cells. and heterodimerization with MTMR5. Furthermore, the K598 MTMR2 was immunoprecipitated by using anti-GFP antibody, and A605 residues are also important for normal coiled-coil and the associated MTMR5 was analyzed by Western blot function. Interestingly, all MTM family members have a putative analysis with anti-MTMR5 antibody. As shown in Fig. 2B Upper, coiled-coil domain in their C-terminal regions. Sequences within

4494 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0431052100 Kim et al. Downloaded by guest on September 28, 2021 Fig. 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 domains͞motifs 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).

their coiled-coil domains may drive specificity for protein– MTMR2 Localization Is Regulated Through Its Interaction with MTMR5. protein interactions. Our results raise the possibility that mul- The finding that MTMR2 interacts with MTMR5 raises the tiple inactive MTM family members may interact with other possibility that a catalytically inactive MTMR5 may regulate the active MTMs. activity of MTMR2. To examine the effect of MTMR5 on MTMR2 enzymatic activity, equal molar amounts of purified recombinant GST-tagged MTMR5 and His-tagged MTMR2 BIOCHEMISTRY were mixed and tested for lipid phosphatase activity by using PI(3)P and phosphatidylinositol 3,5-bisphosphate as substrates. MTMR5 consistently increased MTMR2 phosphatase activity 4.6- and 3.4-fold for PI(3)P and phosphatidylinositol 3,5- bisphosphate, respectively (data not shown). Although a 3- to 5-fold change in MTMR2 catalytic activity may seem modest, the effect of MTMR5 on MTMR2 catalytic activity may be more pronounced under conditions that favor a MTMR2͞MTMR5 heterointeraction, because we were unable to determine the ratio of MTMR2͞MTMR2 homodimer to MTMR2͞MTMR5 heterodimer present in our assay. We next examined the effect of MTMR5 on regulating the subcellular distribution of MTMR2. COS-1 cells were cotrans- Fig. 3. MTMR2 point mutants diminish its homo-͞heterodimerization. fected with EGFP-tagged MTMR2 expression vectors (WT or (A) Sequence alignment of coiled-coil domain of MTM1 and MTMR2. ⌬coil) and FLAG-tagged MTMR5 expression vectors (WT, Gray boxes represent conserved amino acids. The heptad repeat is denoted ⌬PH, or ⌬coil). As shown in Fig. 4 A and B, MTMR2 (WT) and with the letters a– g, with leucine residues residing at the d position. (B and MTMR5 (WT) were colocalized in the cytoplasmic region with C) 293 cells were transfected with FLAG-tagged MTMR2 point mutant strong perinuclear staining. In contrast, MTMR2-⌬coil did not expression vectors alone (B) or combined with EGFP-MTMR2 (WT) expres- colocalize with WT MTMR5, showing a diffuse staining sion vectors (C) as indicated. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation by using anti-FLAG antibody fol- pattern throughout the whole cell, indicating that the coiled- lowed by Western blot analysis by using anti-MTMR5 (B) or anti-GFP coil domain of MTMR2 is important for its subcellular local- (C) antibody. The expression level of proteins in the transfected cells ization (Fig. 4 C and D). We next examined whether impairing was monitored by Western blot analysis with anti-FLAG and͞or anti-GFP the MTMR5 ability to associate with MTMR2 would affect the antibody. subcellular localization of WT MTMR2. MTMR5-⌬PH, a

Kim et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4495 Downloaded by guest on September 28, 2021 subcellular pools of PI(3)P (11). Here we demonstrate that MTMR2 can be targeted to a specific subcellular localization through a heterointeraction with MTMR5. Therefore, we propose that heterodimerization is a mechanism by which MTMR2 locates its specific substrates. Among 13 known MTM family members, 5 are catalytically inactive. A potential role in substrate trapping or docking has been proposed (25, 27), but the exact role of the inactive phosphatase members is still unknown. This study signifies that differential binding partners may be a mechanism to assign different biological roles to MTM1 and MTMR2. Recently, Nandurkar et al. (36) isolated and characterized 3-PAP (MTMR12), another catalytically inactive member of the MTM family. They reported that, although recombinant 3-PAP didn’t have enzymatic activity in vitro, immunoprecipitated 3-PAP from human platelets had PI(3)P and phosphatidylinositol 3,4-bisphosphate 3-phosphatase activity, suggesting that 3-PAP is in a complex with an active enzyme in vivo. However, no 3-PAP binding partner was identified in that study. During the preparation of this manuscript, Zerres and colleagues (37) identified a MTMR5 (sbf1)-related gene, which Fig. 4. MTMR5 regulates the subcellular localization of MTMR2. COS-1 cells they refer to as sbf2. Interestingly, mutations in this gene were cotransfected with EGFP-MTMR2 (WT or ⌬-coil) in combination with produce another form of Charcot–Marie-Tooth disease, type FLAG-MTMR5 (WT, ⌬-PH, or ⌬-coil) expression vectors. Thirty hours after 4B2, an autosomal recessive disease that displays focally folded transfection, cells were fixed and immunostained by using anti-FLAG anti- myelin sheaths. Sbf2 shares 59% amino acid sequence identity body. Green fluorescent staining for EGFP-MTMR2 (WT and ⌬-coil) is shown in with MTMR5. Mutations in MTMR2 have been shown to be A, C, E, and G. Red fluorescent staining for Cy3-conjugated anti-mouse sec- responsible for type 4B1 Charcot–Marie-Tooth disease, which ⌬ ⌬ ondary antibody bound to the FLAG-MTMR5 (WT, -PH, and -coil) is shown also displays focally folded myelin sheaths (23, 24). How do in B, D, F, and H. mutations in a dead phosphatase (sbf2) and in MTMR2 both result in a disease that shares a common phenotype? Our variant that is able to interact with MTMR2, was fully capable results have shown that MTMR2 associates with MTMR5. This of colocalizing with MTMR2 (Fig. 4 E and F). However, association effects MTMR2 activity as well as the subcellular MTMR5-⌬coil, a variant that cannot interact with MTMR2, localization of the active phosphatase. The data described in this paper suggest that MTMR2 may also associate with lead to a diffused subcellular distribution of WT MTMR2 (Fig. sbf2, and the absence of an intact sbf2 gene product may result 4 G and H). A small fraction of MTMR2 still showed the in disease by effecting the activity and͞or subcellular local- perinuclear staining pattern, presumably because of the pres- ization of MTMR2. It will now be interesting and important ence of endogenous MTMR5. Subcellular localization of to determine whether other active and inactive members of MTMR5 was not affected by either the PH domain or the the MTM family interact to achieve their specific biological coiled-coil domain deletion. Our data indicate that, through function. their coiled-coil domain interaction, MTMR5 can regulate the subcellular localization of MTMR2. Although MTM1 could not form a heterodimer with MTMR5, MTM1-⌬coil also We thank Dr. C. D. Worby and Dr. G. S. Taylor for critical review of this manuscript and Dr. S.-G. Ahn for helpful discussion. We also showed diffused staining pattern throughout the whole cell, thank members of the PTP group in Dixon laboratory for valuable suggesting the coiled-coil domain of MTM1 is also important discussions. This work was supported in part by a grant from the for its subcellular localization (data not shown). In a previous National Institutes of Health and the Walther Cancer Institute study, we showed that MTM1 and MTMR2 use different (to J.E.D.).

1. Laporte, J., Blondeau, F., Buj-Bello, A. & Mandel, J.-L. (2001) Trends Genet. 12. Laporte, J., Liaubet, L., Blondeau, F., Tronchere, H., Mandel, J.-L. & 17, 221–228. Payrastre, B. (2002) Biochem. Biophys. Res. Commun. 291, 305–312. 2. Maehama, T., Taylor, G. S. & Dixon, J. E. (2001) Annu. Rev. Biochem. 70, 13. Berger, P., Bonneick, S., Willi, S., Wymann, M. & Suter, U. (2002) Hum. Mol. 247–279. Genet. 11, 1569–1579. 3. Wishart, M. J., Taylor, G. S., Slama, J. T. & Dixon, J. E. (2001) Curr. Opin. Cell 14. Gaullier, J. M., Simonsen, A., D’Arrigo, A., Bremnes, B., Stenmark, H. & Biol. 13, 172–181. Aasland, R. (1998) Nature 394, 432–433. 4. Denu, J. M. & Dixon, J. E. (1998) Curr. Opin. Chem. Biol. 2, 633–641. 15. Dowler, S., Currie, R. A., Campbell, D. G., Deak, M., Kular, G., Downes, C. P. 5. Cui, X., De Vivo, I., Slany, R., Miyamoto, A., Firestein, R. & Cleary, M. L. & Alessi, D. R. (2000) Biochem. J. 351, 19–31. (1998) Nat. Genet. 18, 331–337. 16. Xu, Y., Hortsman, H., Seet, L., Wong, S. H. & Hong, W. (2001) Nat. Cell Biol. 6. Laporte, J., Blondeau, F., Buj-Bello, A., Tentler, D., Kretz, C., Dahl, N. & 3, 658–666. Mandel, J.-L. (1998) Hum. Mol. Genet. 7, 1703–1712. 17. Kanai, F., Liu, H., Field, S. J., Akbary, H., Matsuo, T., Brown, G. E., Cantley, 7. Taylor, G. S., Maehama, T. & Dixon, J. E. (2000) Proc. Natl. Acad. Sci. USA L. C. & Yaffe, M. B. (2001) Nat. Cell Biol. 3, 675–678. 97, 8910–8915. 18. Laporte, J., Hu, L. J., Kretz, C., Mandel, J.-L., Kioschis, P., Coy, J. F., Klauck, 8. Blondeau, F., Laporte, J., Bodin, S., Superti-Furga, G., Payrastre, B. & Mandel, S. M., Poustka, A. & Dahl, N. (1996) Nat. Genet. 13, 175–182. J.-L. (2000) Hum. Mol. Genet. 9, 2223–2229. 19. Laprote, J., Biancalana, V., Tanner, S. M., Kress, W., Schneider, V., Wallgren- 9. Walker, D. M., Urbe, S., Dove, S. K., Tenza, D., Raposo, G. & Clague, M. J. Pettersson, C., Herger, F., Buj-Bello, A., Blondeau, F., Liechti-Gallati, S., et al. (2001) Curr. Biol. 11, 1600–1605. (2000) Hum. Mutat. 15, 393–409. 10. Zhao, R., Qi, Y., Chen, J. & Zhao, Z. J. (2001) Exp. Cell Res. 265, 20. Spiro, A. J., Shy, G. M. & Gonatas, N. K. (1966) Arch. Neurol. 14, 1–14. 329–338. 21. Bolino, A., Muglia, M., Conforti, F. L., LeGuern, E., Salih, M. A. M., Georgiou, 11. Kim, S.-A, Taylor, G. S., Torgersen, K. M. & Dixon, J. E. (2002) J. Biol. Chem. D.-M., Christodoulou, K., Hausmanowa-Petrusewicz, I., Mandich, P., Sche- 277, 4526–4531. none, A., et al. (2000) Nat. Genet. 25, 17–19.

4496 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0431052100 Kim et al. Downloaded by guest on September 28, 2021 22. Houlden, H., King, R. H., Wood, N. W., Thomas, P. K. & Reilly, M. M. (2001) 31. Vacratsis, P. O., Phinney, B. S., Gage, D. A. & Gallo, K. A. (2002) Biochemistry Brain 124, 907–915. 41, 5613–5624. 23. Bolino, A., Brancolini, V., Bono, F., Bruni, A., Gambardella, A., Romeo, G., 32. Maehama, T., Taylor, G. S., Slama, J. T. & Dixon, J. E. (2000) Anal. Biochem. Quattrone, A. & Devoto, M. (1996) Hum. Mol. Genet. 5, 1051–1054. 279, 248–250. 24. Gambardella, A., Muglia, M. & Quattrone, A. (1997) Brain 120, 2113– 33. Taylor, G. S., Liu, Y., Baskerville, C. & Charbonneau, H. (1997) J. Biol. Chem. 2115. 272, 24054–24063. 25. Flint, A. J., Tiganis, T., Barford, D. & Tonks, N. K. (1997) Proc. Natl. Acad. 34. Schultz, J., Milpetz, F., Bork, P. & Ponting, C. P. (1998) Proc. Natl. Acad. Sci. Sci. USA 94, 1680–1685. USA 95, 5857–5864. 26. Hunter, T. (1998) Nat. Genet. 18, 303–305. 35. Lupas, A., Van Dyke, M. & Stock, J. (1991) Science 252, 1162–1164. 27. Wishart, M. J. & Dixon, J. E. (1998) Trends Biochem. Sci. 23, 36. Nandurkar, H. H., Caldwell, K. K., Whisstock, J. C., Layton, M. J., Gaudet, 301–306. E. A., Norris, F. A., Majerus, P. W. & Mitchell, C. A. (2001) Proc. Natl. Acad. 28. Firestein, R., Nagy, P. L., Daly, M., Huie, P., Conti, M. & Cleary, M. L. (2002) Sci. USA 98, 9499–9504. J. Clin. Invest. 109, 1165–1172. 37. Senderek, J., Bergmann, C., Weber, S., Ketelsen, U.-P., Schorle, H., Rudnik- 29. Michael, S. F. (1994) BioTechniques 16, 410–412. Scho¨neborn, S., Bu¨ttner, R., Buchheim, E. & Zerres, K. (2003) Hum. Mol. 30. Firestein, R. & Cleary, M. L. (2001) J. Cell Sci. 114, 2921–2927. Genet. 12, 349–356. BIOCHEMISTRY

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