# 2007 The Authors Journal compilation # 2007 Blackwell Publishing Ltd Traffic 2007; 8: 1052–1067 Blackwell Munksgaard doi: 10.1111/j.1600-0854.2007.00586.x Myotubularin Lipid Phosphatase Binds the hVPS15/ hVPS34 Lipid Kinase Complex on Endosomes Canhong Cao1, Jocelyn Laporte2,3,4,5,6, Received 14 July 2006, revised and accepted for publica- Jonathan M. Backer7, Angela Wandinger- tion 20 April 2007, uncorrected manuscript published online 24 April 2007 Ness1,* and Mary-Pat Stein8 1Molecular Trafficking Laboratory, Department of Myotubularins define a diverse family of lipid phospha- Pathology, University of New Mexico School of tases that when mutated result in severe human diseases Medicine, Albuquerque, NM 87131, USA 2 affecting different tissues such as muscle in X-linked Department of Molecular Pathology, IGBMC (Institut de myotubular myopathy (MTM1)(1,2) and the peripheral Ge´ ne´ tique et de Biologie Mole´ culaire et Cellulaire), Illkirch F-67400, France nervous system in Charcot-Marie-Tooth diseases type 3Inserm, U596, Illkirch F-67400, France 4B1(3) and type 4B2 (2,4). The MTM family consists of 4CNRS, UMR7104, Illkirch F-67400, France 14 highly conserved dual-specificity protein tyrosine phos- 5Universite´ Louis Pasteur, Strasbourg F-67000, France phatase (PTP)-like enzymes: eight are catalytically active 6 Colle` ge de France, Chaire de Ge´ ne´ tique Humaine, and six contain mutations in their HCX5R PTP catalytic Illkirch F-67400, France motif, rendering them phosphatase dead (5,6). Although 7 Department of Molecular Pharmacology, Albert Einstein homologues do not compensate for individual family College of Medicine, Bronx, NY 10461, USA members, cooperation between catalytically active and 8Department of Biology, California State University Northridge, Northridge, CA 91330, USA inactive myotubularins has been suggested to occur *Corresponding author: Angela Wandinger-Ness, (2,7–9). In addition to a PTP domain, each family member [email protected] has a Suvar 3-9, Enhancer of zeste, Trithorax-interacting domain (10). Most members also have a coiled-coil domain that mediates homodimerization and/or heterodimerization (11,12), and one or more phosphoinositide-binding do- Myotubularins constitute a ubiquitous family of phos- mains that facilitate myotubularin membrane localization phatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular (11,13). The human myotubularin family appears to be myopathy 1 (MTM1)] PI 3-phosphatase is shown associ- expressed in all tissues with the exception of hMTMR7, ated with early and late endosomes. Loss of endosomal which is only expressed in brain (14). Thus, differential phosphatidylinositol 3-phosphate [PI(3)P] upon overex- expression levels, intracellular localization or activation of pression of wild-type MTM1, but not a phosphatase-dead distinct myotubularin enzymes may account for different MTM1C375S mutant, resulted in altered early and late disease states observed when closely related myotubularin endosomal PI(3)P levels and rapid depletion of early family members are mutated. Understanding how mutant endosome antigen-1. Membrane-bound MTM1 was myotubularins cause disease necessitates a more detailed directly complexed to the hVPS15/hVPS34 [vacuolar pro- tein sorting (VPS)] PI 3-kinase complex with binding understanding of their cellular localizations, functions and mediated by the WD40 domain of the hVPS15 (p150) interacting partners. adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P2 binding. The WD40 Although first described as dual-specificity PTP enzymes, domain of hVPS15 also constitutes the binding site for the primary substrates for myotubularins in yeast and Rab7 and, as shown previously, contributes to Rab5 mammalian cells are the phosphoinositides PI(3)P (15,16) binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex and PI(3,5)P (17–19). These phosphoinositide species are forms mutually exclusive complexes with the Rab 2 localized to early and late endosomes and play important GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab roles in membrane trafficking (20,21). In particular, PI(3)P is GTPases together with MTM1 likely serve as molecular required for the recruitment of factors containing Fab1, switches for controlling the sequential synthesis and YOTB/ZK632.12, Vac1 and early endosome antigen-1 degradation of endosomal PI(3)P. Normal levels of endo- (EEA1) FYVE or phox (PX) domains (22–25). These factors somal PI(3)P and PI(3,5)P2 are crucial for both endosomal include tethering factors (EEA1), phosphoinositide kinases morphology and function, suggesting that disruption of (PIKfyve/Fab1p) and intralumenal vesicle forming machin- endosomal sorting and trafficking in skeletal muscle ery (hepatocyte growth factor-regulated tyrosine kinase when MTM1 is mutated may be a key factor in pre- substrate, Hrs/Vps27p) recruited to early and late endo- cipitating X-linked MTM. somes (26,27). The binding of FYVE/PX domain proteins Key words: Charcot-Marie-Tooth neuropathy, endocyto- to PI(3)P-containing endosomes provides a platform for sis, phosphatidylinositol 3-phosphatase or 3-kinase, Rab the recruitment and assembly of multiprotein complexes GTPase, X-linked myotubular myopathy that promote endosomal tethering, docking, fusion and 1052 www.traffic.dk MTM1 Binds hVPS15/hVPS34 PI 3-Kinase membrane remodeling, thereby facilitating endocytic hVPS34 complex. In addition, we demonstrate that both transport and receptor sorting. This has been demon- Rab7 and MTM1 bind to hVPS15 (formerly called p150), strated most clearly by numerous studies on the sorting the hVPS34 adapter molecule (42) that regulates hVPS34 and degradation of epidermal growth factor receptor activity through its interactions with Rab5 (38) and Rab7 (EGFR), which is highly dependent on PI(3)P and (32,41). The WD40 domain of hVPS15 mediates binding to PI(3,5)P2 (13,28,29). Increased degradation of PI(3)P and Rab5, Rab7 and MTM1. Consequently, binding of the PI(3,5)P2 by overexpression of MTM1 has been shown to hVPS15/hVPS34 complex to the Rab GTPases and cause aberrant endosomal sorting and result in a profound MTM1 is mutually exclusive. The data suggest that tight inhibition of epidermal growth factor (EGF)-stimulated regulation and rapid turnover of the phosphoinositide- receptor degradation (13). Thus, myotubularins play an mediated signals may be accomplished through the cou- important role in integrating cellular phosphoinositide pling of proteins involved in the synthesis and turnover of regulation, membrane trafficking and growth control. PI(3)P on endosomes. In mammalian cells, early and late endosomal PI(3)P are generated by the PI 3-kinase hVPS34 [vacuolar protein Results sorting (VPS)] (30–33), and the PI(3)P 5-kinase PIKfyve generates PI(3,5)P2 from PI(3)P on late endosomes (34). MTM1 is recruited to Rab5- and Rab7-positive early These phosphoinositide species are thought to be short and late endosomes lived because of rapid modification by kinases, dephos- Overexpressed MTM1 has been observed at the plasma phorylation by phosphatases such as the myotubularins or membrane and diffusely dispersed in the cytosol (15,16) degradation by lipases. In yeast, genetic studies demon- and has been shown to diminish early endosomal PI(3)P strate that PI(3)P is transported to vacuoles (the mamma- (43). Recently, however, EGF-dependent recruitment of lian equivalent of lysosomes), where it is either converted overexpressed MTM1 to kinetically defined late endo- into PI(3,5)P2 by Fab1p (35) or degraded by vacuolar somes has been reported (13). As a first step toward hydrolases (36). In mammalian cells, conversion of PI(3)P assessing the endosomal localization and recruitment of to PI(3,5)P2 by PIKfyve on late endosomes is thought to MTM1 more precisely, endogenous and overexpressed trigger the inward invagination of intraluminal vesicles into MTM1 and a phosphatase-dead mutant of MTM1 multivesicular bodies (MVB) and to facilitate sequestration (MTM1C375S) were localized relative to specific early of cargo such as EGFR into MVB (13,37). Hence, balanced and late endosomal markers. synthesis and consumption of PI(3)P and PI(3,5)P2 species on endosomal compartments ensures proper and efficient The endosomal distribution of endogenous MTM1 was endocytic transport. evaluated relative to markers of early and late endosomes (Figure 1A). To reveal the membrane-bound pool of The recruitment and activity of the hVPS34 PI 3-kinase on MTM1, cells were saponin extracted, a method well early and late endosomes depends on the upstream ac- known to preserve membrane ultrastructure while extract- tivation of the small GTPases Rab5 and Rab7, respectively ing cytosolic content (44,45). Endogenous MTM1 was (32,38). Through their organelle-specific localization and consistently observed outlining early endosomal mem- tightly regulated nucleotide binding and hydrolysis cycle, branes that were positive for Rab5. The Pearson’s corre- Rab GTPases are ideally poised as pivotal regulators of lation coefficient (range À1toþ1) for MTM1 and Rab5 was vesicular trafficking (reviewed in 64). Thus, a delicate 0.8 Æ 0.03, indicating a high degree of colocalization. The balance between Rab GTPases, lipid kinases and lipid incomplete overlap between EEA1 and MTM1 may reflect phosphatases must exist to regulate endosomal transport. localization of EEA1 to discrete early endosomal mem- Indirect evidence exists for the