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Identification of Myotubularin As the Lipid Phosphatase Catalytic Subunit Associated with the 3-Phosphatase Adapter Protein, 3-PAP

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Identification of Myotubularin As the Lipid Phosphatase Catalytic Subunit Associated with the 3-Phosphatase Adapter Protein, 3-PAP Identification of myotubularin as the lipid phosphatase catalytic subunit associated with the 3-phosphatase adapter protein, 3-PAP Harshal H. Nandurkar*†, Meredith Layton‡, Jocelyn Laporte§, Carly Selan*, Lisa Corcoran*¶, Kevin K. Caldwellʈ, Yasuhiro Mochizuki**, Philip W. Majerus**, and Christina A. Mitchell¶ *St. Vincent’s Hospital, 3065 Melbourne, Australia; ‡Ludwig Institute of Medical Research, 3050 Parkville, Australia; §Institute of Genetics and Molecular and Cellular Biology, 67404 Illkirch Cedex, France; ¶Department of Biochemistry and Molecular Biology, Monash University, 3068 Clayton, Australia; ʈDepartment of Neuroscience, University of New Mexico, Albuquerque, NM 87131; and **Department of Haematology, Washington University Medical School, St. Louis, MO 63110 Contributed by Philip W. Majerus, May 21, 2003 Myotubularin is a dual-specific phosphatase that dephosphory- syndrome, a hereditary demyelinating peripheral neuropathy lates phosphatidylinositol 3-phosphate and phosphatidylinositol (17, 18). (3,5)-bisphosphate. Mutations in myotubularin result in the human The catalytically inactive myotubularin family members in- disease X-linked myotubular myopathy, characterized by persis- clude SET-binding factor 1 (MTMR5), LIP-STYX (MTMR9), tence of muscle fibers that retain an immature phenotype. We have MTMR10, the 3-phosphatase adapter protein (3-PAP) previously reported the identification of the 3-phosphatase (MTMR12), and MTMR13 (SET-binding factor 2) (7, 14–16, 19, adapter protein (3-PAP), a catalytically inactive member of the 20). The function of these catalytically inactive phosphatases is myotubularin gene family, which coprecipitates lipid phosphati- unknown. Very recently, mutations in MTMR13 have been dylinositol 3-phosphate-3-phosphatase activity from lysates of described in type 4B2 Charcot Marie Tooth syndrome (19, 20). human platelets. We have now identified myotubularin as the Also, male mice deficient in SET-binding factor 1 are azoosper- catalytically active 3-phosphatase subunit interacting with 3-PAP. matic and infertile (21). We originally purified 3-PAP from rat A 65-kDa polypeptide, coprecipitating with endogenous 3-PAP, brain as a heterodimer with an unidentified 65-kDa PtdIns(3)P was purified from SDS͞PAGE, subjected to trypsin digestion, and 3-phosphatase (22). The human 3-PAP cDNA had sequence and analyzed by collision-induced dissociation tandem MS. Three pep- domain homology with myotubularin, but lacked the HCX5R tides derived from human myotubularin were identified. Associa- catalytic motif (23). Recombinant 3-PAP did not dephosphor- tion between 3-PAP and myotubularin was confirmed by reciprocal ylate PtdIns(3)P; however, 3-PAP immunoprecipitates from coimmunoprecipitation of both endogenous and recombinant pro- human platelet cytosol exhibited PtdIns(3)P 3-phosphatase ac- teins expressed in K562 cells. Recombinant myotubularin localized tivity, suggesting 3-PAP functions as an adapter subunit to an to the plasma membrane, causing extensive filopodia formation. unidentified PtdIns(3)P 3-phosphatase (23). In this study we have identified myotubularin as a catalytic However, coexpression of 3-PAP with myotubularin led to atten- subunit that complexes with 3-PAP. Furthermore, 3-PAP de- uation of the plasma membrane phenotype, associated with myo- termines myotubularin cytosolic localization, and thereby alters tubularin relocalization to the cytosol. Collectively these studies the phenotype of myotubularin-overexpressing cells. indicate 3-PAP functions as an ‘‘adapter’’ for myotubularin, regu- lating myotubularin intracellular location and thereby altering the Materials and Methods phenotype resulting from myotubularin overexpression. Materials. Restriction and DNA-modifying enzymes were ob- tained from Promega. Antibodies against myotubularin have hosphoinositide 3-kinase-derived molecules regulate diverse been described (24). Antibody to the FLAG epitope (mAb Psignaling functions including vesicular trafficking, actin cy- KM5–1C7) was obtained from the Walter and Eliza Hall Insti- toskeletal rearrangement, apoptosis, and cell proliferation (1, 2). tute of Medical Research (Melbourne). mAb to the hemagglu- The signaling molecule phosphatidylinositol 3-phosphate tinin (HA) epitope used to probe immunoblots was obtained [PtdIns(3)P] binds and localizes FYVE, Phox, and pleckstrin from Silenus (Novi, MI). Polyclonal antibody to the HA epitope homology domain-containing proteins to specific subcellular used in microscopy was obtained from Upstate Biotechnology compartments such as early and late endosomes and thereby (Lake Placid, NY). All fluorochrome-tagged secondary anti- regulates endosomal and other vesicular trafficking pathways bodies (Alexa Fluor) were supplied by Molecular Probes, and (3–5). horseradish peroxidase-tagged secondary antibodies for immu- Myotubularin is a dual-specificity protein tyrosine phos- noblot analysis were obtained from DAKO. The pCGN vector phatase-like enzyme, which contains a HCX5R catalytic motif was a gift from Tony Tiganis (Monash University), pEFBOS- and functions as a PtdIns(3)P and phosphatidylinositol 3,5- FLAG vector was a gift from Tracey Willson (Walter and Eliza bisphosphate [PtdIns(3,5)P2] 3-phosphatase (6–11, 12). The Hall Institute of Medical Research), and the myotubularin and myotubularin gene locus, MTM1, is mutated in X-linked myo- MTMR2 cDNA were from G. S. Taylor (University of Michigan tubular myopathy, a severe congenital disorder characterized by Medical School, Ann Arbor). All other reagents were from neonatal generalized muscle weakness, hypotonia, and death Sigma-Aldrich unless otherwise stated. early in life (13). Myotubularin defines the largest family of lipid phosphatases in the human genome, which includes eight cata- Cloning of MTMR6 cDNA. The MTMR6 cDNA was amplified by lytically active enzymes, and in addition six putative inactive PCR, using the human brain cDNA library (Marathon Ready homologues of unknown function, which contain mutations within the HCX5R catalytic motif (14–16). Mutations in the Abbreviations: PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3,5)P2, phosphatidyl- myotubularin-related gene MTMR2, which encodes a PtdIns(3)P inositol 3,5-bisphosphate; 3-PAP, 3-phosphatase adapter protein; HA, hemagglutinin; SID, and PtdIns(3,5)P2 3-phosphatase, are responsible for the neu- SET-interacting domain. rodegenerative disorder, type 4B1 Charcot Marie Tooth †To whom correspondence should be addressed. E-mail: [email protected]. 8660–8665 ͉ PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1033097100 Downloaded by guest on September 23, 2021 troporation and harvested 48 h later. Lysates were prepared, and immunoprecipitation was as described above. Epitope-tagged recombinant proteins were immunoprecipitated with 1–2 ␮gof the appropriate anti-HA or anti-FLAG antibody. All immuno- blots were probed with goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase and detected by enhanced chemiluminescent substrate (Pierce). Recombinant FLAG-tagged MTM1, MTMR2, and HA- tagged MTMR6 were coexpressed with Myc-tagged 3-PAP in COS-7 cells. Myc-tagged 3-PAP was immunoprecipitated with anti-Myc polyclonal antibody and probed by using anti-Flag antibody for MTM1 and MTMR2 and anti-HA antibody for MTMR6. Immunoprecipitates of Myc-3-PAP were also probed with anti-Myc antibody to ensure equal loading of recombinant 3-PAP. Identification of Myotubularin in the Immunoprecipitates of Endog- enous 3-PAP. Immunoprecipitates from 5 ϫ 108 K562 cells (per- formed in batches and pooled), using 250 ␮g of anti-3-PAP antibody and 1,000 ␮l of slurry of protein A-Sepharose in Fig. 1. Immunoprecipitation of 3-PAP and a 65-kDa polypeptide. (A) Mod- ⅐ ͞ ular structure of 3-PAP and 3-PAP⌬SID. The extent of the myotubularin Tris NaCl, were separated by 1D SDS PAGE (7.5% acrylamide) homology region and the SID is shown. The box identified by a star represents and stained with Coomassie blue R250. The polypeptide mi- the mutated catalytic motif. Amino acids 24–40 represent the immunogenic grating at 65 kDa was excised and digested in situ with 0.5 ␮gof peptide used to raise the polyclonal rabbit anti-3-PAP antibody. The amino trypsin (25). Generated peptides were resolved by using capillary acids deleted in 3-PAP⌬SID variant are shown. (B) Proteins were immunopre- reversed-phase HPLC (26) and identified by collision-induced cipitated (IP) from 107 K562 cells, using 5 ␮g of either anti-3-PAP antibody dissociation tandem MS using a quadrupole ion-trap mass (anti-3-PAP) or nonimmune sera (NI), and examined by immunoblot (WB) spectrometer (model LCQ, Finnigan-MAT, San Jose, CA) analysis probed with the anti-3-PAP antibody. The migration of 3-PAP and Ig equipped with electrospray ionization (27). The sequences of heavy chain (Ig) is marked by arrows. MW indicates the migration of molecular mass markers (kDa). (C) Immunoprecipitates prepared as above were pooled uninterpreted collision-induced dissociation spectra were iden- and separated by SDS͞PAGE and visualized by silver stain. The migration of the tified by using the SEQUEST algorithm and confirmed by manual de 65-kDa polypeptide that coimmunoprecipitates with 3-PAP is indicated. novo interpretation of their b- and y-type production series (28). PtdIns(3)P 3-Phosphatase Assays. PtdIns(3)32P was generated as cDNA kit, CLONTECH) as a template and ligated into a described (23). Immunoprecipitates prepared from parental pCMV5-HA expression
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