Receptor mediated endocytosis 8 is a novel PI(3)P binding protein regulated by myotubularin-related 2

RME8 and EGFR colocalize by fluorescence microscopy (View interaction) The phosphatidylinositol phosphate (PIP) isoforms PI(3)P and PI(3,5)P2 are known to serve as membrane targeting ligands for proteins that are essential for cellular membrane trafficking processes. These lipid–protein interactions function in translocating signaling proteins to discrete membrane locations where they can properly respond to extracellular stimuli [1]. The signaling pathways are subsequently turned off through the dephosphorylation of the PIPs. The MTMR family of lipid phosphatases, composed of active and inactive subgroups, represents the largest protein tyrosine phosphatase (PTP) subfamily conserved from yeast to humans [2, 3]. Initially, MTM1 was the first family member shown to dephosphorylate the D3 position of PI(3)P in vitro and in vivo [4, 5]. Subsequently, all active members tested have been shown to also be PI(3)P phosphatases [6, 7]. Furthermore, there is accumulating evidence that MTMs can also utilize PI(3,5)P2 as a physiologic substrate [6, 8, 9]. Therefore, MTMs are thought to antagonize effector molecules that utilize PI(3)P or PI(3,5)P2 as targeting ligands and/or allosteric activators. Mutations in mtmr2, on chromosome 11q22, have been shown to cause the neurodegenerative disorder, Charcot-Marie-tooth disease 4B1 (CMT4B1) [10]. CMT4B1 is an autosomal recessive aggressive form of CMT, characterized by abnormally folded myelin sheaths, inadequate nerve signaling to muscles, and eventual muscle weakness and atrophy [11]. While the pathophysiological consequence resulting from the loss of myotubularin related protein 2 (MTMR2) function is well established, how MTMR2 participates in trafficking events remains poorly understood. Recently we have characterized an N-terminal MTMR2 phosphorylation site at position Ser58 that dramatically regulates MTMR2 endosomal localization and thus access to its lipid substrates [12]. A phosphorylation deficient variant (MTMR2 S58A) displays strong endosomal localization and an enhanced ability to deplete PI(3)P from endosomal vesicles signifying that reversible phosphorylation is a critical mechanism regulating the activities of MTMR2. The biological pathways affected by MTMR proteins remain inadequately characterized. This task has proven difficult due to the poor understanding of PIP binding domains that specifically interact with the PIP isoforms that MTMRs target [1]. In this study, we utilized a proteomic approach using immobilized PIP isoforms to identify novel PIP binding proteins. Herein, we describe the identification of receptor mediated endocytosis 8 (RME-8) as a novel PI(3)P binding protein whose lipid binding activity is affected by MTMR2. Immobilized PIP isoforms (Echelon) were prepared for affinity chromatography according to the manufacturer's protocol. RT4-D6P2T Schwann cells (6 × 107) were grown to 80% confluency, lysed and the soluble lysate filtered. The cellular lysates was pre-cleared with PI control beads followed by the loading of 10 mg of total protein to either PIP, PI(3)P, PI(3,5)P2, or PI(5)P resin. The lipid pulldown was performed by batch method overnight at 4 °C. Following rigorous washing conditions the bound proteins were eluted in SDS–PAGE loading buffer and analyzed by SDS–PAGE and silver staining. Protein bands of interest were excised and in-gel digested with trypsin. The extracted pools of tryptic peptides were then analyzed by matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) for protein identification as described previously [13]. HeLa and HEK293 cells were utilized for their reproducible transfection efficiency and were maintained in DMEM/ F-12 supplemented with 10% FBS, 1% penicillin/streptomycin and maintained at 37 °C with 5% CO2. Cells were transiently transfected with expression vectors encoding FLAG tagged-MTMR2 S58A [12] and GFP-RME-8 variants [14] using FuGene HD Transfection Reagent (Roche) according to the manufacturer's protocol. The DsRed-FYVE construct was created by PCR amplification of the sequence encoding the FYVE domain of EEA1 followed by cloning into the pDsRed expression vector (Clonetech). Where indicated, wortmannin treatment (1 μM) was for 1 h at 37 °C. HEK293 cells were transiently transfected with GFP-RME-8 variants for 24 h and lysed in modified RIPA buffer (50 mM Tris–HCl pH 7.4, 1% Nonidet P-40, 76 mM NaCl, 2 mM EGTA, 10% Glycerol) supplemented with protease inhibitors PMSF (1 mM) and aprotinin (1 mM). Cell lysates were incubated with PI(3)P, PI(3,5)P2 and PIP conjugated resin (Echelon Research Laboratories) overnight at 4 °C. The samples were washed 3 times with 10 mM HEPES pH 7.4, 150 mM NaCl, 0.25% NP-40 and resuspended in SDS–PAGE loading dye. The protein samples were separated on an 8% SDS–PAGE, transferred to a PVDF membrane and immunoblotted with goat anti-GFP (Rockland) as the primary antibody and rabbit-anti goat HRP (Rockland) as the secondary antibody. Proteins were visualized using Super Signal West Femto reagent (Thermo scientific). Following transient transfection, Hela cells were fixed at room temperature with 3.7% paraformaldehyde in PBS. Cells were then permeabilized with 0.15% Triton X-100 in PBS at room temperature for 2 min and blocked for 1 h in 5% BSA (Sigma). Cells were incubated with mouse anti-FLAG antibody (Sigma–Aldrich) or rabbit anti-EGFR (Santa Cruz Biotechnology) in TBST. Following washing, cells were incubated with either Alexa 568 goat anti-mouse, Alexa 350 donkey anti-rabbit (Invitrogen) or fluorescein goat anti-rabbit (Vector laboratories) secondary antibodies. All the incubations were performed at room temperature for 1 h followed by three 5 min washes in TBST. Hoechst 33342 (Molecular Probes) was used to stain the nuclei and Slowfade Antifade kit was used to mount the slides (Molecular Probes) according to the manufacturer's protocol. Images were captured with a Q-imaging CCD camera on a Leica DMIRB microscope using the Northern Eclipse software and Abobe Photoshop 7.0. Knowledge of proteins affected by the lipid phosphatase activity of MTMR2 is poorly defined. To identify putative targets of MTMR2, a pull down assay was performed using disease relevant Schwann cells and conjugated PI(3)P and PI(3,5)P2 beads. Beads conjugated with the enzymatic products of MTMR2, PI and PI(5)P, were also included. The PIP beads were incubated with equal concentrations of cellular lysate from rat RT4-D6P2T Schwann cells followed by extensive washing. Following elution in SDS–PAGE loading dye, the proteins were separated by SDS–PAGE and detected by silver staining (Fig. 1 ). Several protein bands appeared specific to the PIP isoforms including two protein bands observed at molecular weights of approximately 205 and 70 kDa that were specific for PI(3)P and PI(3,5)P2 (indicated by asterisks). Protein bands of interest were excised and in-gel digested with trypsin. The extracted pools of tryptic peptides were then analyzed by MALDI-TOF MS/MS for protein identification. The 205 kDa band was identified as receptor-mediated endocytosis 8 (RME-8) and the 70 kDa band was identified as heat shock cognate 70 (Hsc70) (Fig. 2 ). At least 10 peptides from each trypsin digest were subjected to MS/MS analysis to obtain unambiguous identification. The high sequence coverage was particular important in the case of Hsc70 in order to distinguish from closely related homologues. Notably, the 200 kDa band present in the PI(5)P sample (Fig. 1) was identified as pyruvate decarboxylase (data not shown). The identification of RME-8 and Hsc70 is intriguing since these two proteins in a variety of organisms and cell types have been shown to function in a complex and regulate receptor-mediated and fluid-phase endocytosis [15-17]. Furthermore, Drosophila RME-8 and Hsc70 have been shown to interact in vitro [16], while human RME-8, through its J-domain, has been shown to directly bind to Hsc70 in an ATP dependent manner [17]. It has been demonstrated that RME-8 can localize to membrane vesicles including early endosomes [14, 18]. Our current proteomic results suggest that RME-8 can also interact with the substrates of MTMR2, such as PI(3)P, which are present on membrane vesicles such as endosomes. To test if PI(3)P is required for its endosomal localization, we examined the ability of RME-8 to co-localize with a well characterized reporter for PI(3)P enriched endosomes, DsRed-FYVE [19]. As shown in Fig. 3 A, RME-8 displayed strong co-staining with DsRed-FYVE confirming that RME-8 positive vesicles contain PI(3)P. Cells were then treated with the phosphoinositide 3-kinase (PI(3)K) inhibitor wortmannin to investigate the requirement of PI(3)P for the membrane vesicle localization of RME-8. Wortmannin-induced PI(3)P depletion led to a strong reduction in the endosomal staining pattern of RME-8. Thus, in addition to interacting with PI(3)P beads in vitro, RME-8 associates with PI(3)P containing vesicles in vivo. RME-8 possesses a DnaJ domain in the C-terminal half of the protein that is responsible for interacting with Hsc70 [15, 16]. The C-terminal region also mediates the reported interaction between RME-8 and sorting nexin 1[18]. On the other hand, the N-terminal region has been shown to mediate the membrane localization of RME-8 [14]. Likewise, PIP pull down assays were performed with RME-8 deletion constructs to determine which RME-8 region was responsible for the observed PI(3)P and PI(3,5)P2 binding. The results showed that full length RME-8 and a variant encompassing all but the last 425 amino acids were able to associate with PI(3)P and PI(3,5)P2. In contrast, deletion of the first 453 amino acids completely abolished binding to PI(3)P and PI(3,5)P2 (Fig. 4 ). Taken together, our results strongly suggest that RME-8 is a novel PI(3)P binding protein both in vitro and in cells. We have recently discovered that MTMR2 is highly phosphorylated on Ser58 which sequesters MTMR2 in the cytoplasm and limits access to its lipid substrates [12]. Conversely, a phosphorylation deficient mutant, MTMR2 S58A, is capable of localizing to endosomal vesicles and depleting PI(3)P. Therefore, to address whether the PI(3)P binding ability of RME-8 could be affected by MTMR2, RME-8 was co-expressed with MTMR2 S58A and the integrity of the RME-8 endosomal pattern was analyzed by immunofluorescent microscopy. When overexpressed alone, GFP-RME-8 and MTMR2 S58A both exhibited the characteristic punctate endosomal localization pattern (Fig. 5 A and B, left panels). Strikingly, co-expression of MTMR2 S58A resulted in significant loss of the RME-8 endosomal localization pattern (Fig. 5A, upper right panel), as did treatment with wortmannin (Fig. 5B, right panel). Furthermore, expression of a catalytically inactive variant (MTMR2 S58A.C417S) failed to disrupt the endosomal localization pattern of RME-8 (Fig. 5A, lower right panel), suggesting that the association of RME-8 with PI(3)P rich vesicles can be disrupted by the phosphatase activity of MTMR2. RME-8 has been previously shown to regulate the endosomal sorting of a variety of cargo at the early to late endosome interface, including the epidermal growth factor receptor (EGFR) [14, 17, 18, 20]. Similarly, in HeLa cells that were stimulated with EGF, GFP-RME-8 displayed significant co-localization with EGFR (Fig. 6 A). To ascertain the compartment where RME-8 and EGFR co-localize we utilized triple staining immunofluorescence with the early endosome marker Rab5 (Fig. 6B) and the late endosome/lysosomal marker LAMP1 (Fig. 6C). Although EGFR was found to partially co-localize with both of these markers, vesicles containing EGFR and RME-8 only co-stained with Rab5 and not LAMP1 suggesting that RME-8 co-localizes with EGFR on early endosomes. Meanwhile, expression of MTMR2 S58A disrupted the ability of RME-8 to co-localize with the EGFR (Fig. 6D). Again, this disruption required the phosphatase activity of MTMR2 as RME-8 was capable of co-localizing to EGFR positive vesicles in the presence of the catalytically inactive mutant of MTMR2 (Fig. 6E). Collectively, these results demonstrate that MTMR2 regulates the PI(3)P dependent targeting of RME-8 to EGFR-positive early endosomes. In conclusion, our results have elucidated a novel mechanism for regulating RME-8 endosomal targeting. As RME-8 in a variety of organisms has clearly demonstrated to be a critical regulator of endosomal processing, determining the temporal and spatial details of how PI(3)P regulates the docking of RME-8 to endosomal vesicles will deepen our understanding of the specific role of RME-8 in vesicular trafficking. Seeing that RME-8 is expressed in a variety of mammalian tissues [17], it will also be interesting to investigate if other MTMRs can regulate RME-8 to the same extent as MTMR2. Finally, as CMT4B1 is caused by loss of a functional MTMR2, our findings open the door to examining if RME-8 is overactive and contributes to the demyelination phenotypes in CMT 4B1. This study was funded by an Operating Grant (MOP 89870) from (CIHR) to P.O.V. We thank Norah Franklin and Christopher Bonham for technical assistance.

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