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Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a novel role for ubiquitination in this process. We find that the novel E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the Retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired Retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of Retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and Retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 and reveal novel aspects of retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.
Endosomal protein recycling pathways facilitate the transfer of membrane proteins from early and late endosomes back to the trans-Golgi network (TGN) or plasma membrane (Bonifacino and Rojas, 2006). In doing so, these pathways generally function to prevent lysosomal delivery and degradation of membrane proteins. Endosome-to-Golgi transport, referred to as retrograde transport, is an important cellular process that facilitates the recycling of a variety of proteins, including: sorting receptors (such as CI-M6PR), SNARE membrane fusion proteins, membrane receptors, metabolite transporters, and several proteins that undergo polarized localization/secretion (Bonifacino and Rojas, 2006; Johannes and Popoff, 2008). Importantly, retrograde transport has been implicated in a number of different human pathologies. Endosome-to-Golgi transport is essential for cellular entry of pathogenic toxins, such as Shiga, Cholera, and ricin, as well as viral pathogens such as HIV (Brass et al., 2008; Sandvig and van Deurs, 2005). Furthermore, components of the retrograde transport pathway are downregulated in Alzheimer’s disease and upregulated in cancer (Scott et al., 2009; Small, 2008).
Recent studies have started to define the molecular machinery crucial for different aspects of this process, including cargo recognition, endosomal membrane budding, tubulation, and scission, and vesicle transport, tethering, and fusion at the TGN (Bonifacino and Rojas, 2006; Cullen and Korswagen, 2012). One critical component is the Retromer protein complex that consists of VPS26, VPS29, and VPS35 and functions to recognize retrograde cargo on endosomes (Bonifacino and Hurley, 2008). Another essential factor in retrograde transport is WASH. WASH is a member of the Wiskott-Aldrich syndrome protein (WASP) family consisting of WASP/N-WASP, WAVE, WHAMM, JMY, and WASH (Campellone and Welch, 2010). Like other WASP family members, WASH contains a carboxy-terminal VCA (verprolin homologous or WH2, central hydrophobic, and acidic) motif that binds to actin and the Arp2/3 complex to stimulate actin filament nucleation (Derivery et al., 2009; Duleh and Welch, 2010; Jia et al., 2010; Linardopoulou et al., 2007; Liu et al., 2009). Recent studies have demonstrated that WASH is endosomal-associated, exists in a macromolecular complex termed the WASH Regulatory Complex (SHRC) and functions downstream of the Retromer complex to facilitate endosome-to-Golgi transport (Derivery et al., 2009; Duleh and Welch, 2010; Gomez and Billadeau, 2009; Linardopoulou et al., 2007). Importantly, Retromer and SHRC have been implicated in other endosomal recycling pathways, including endosome-to-plasma membrane sorting of integrins (Zech et al., 2011). SHRC consists of at least five core factors, CCDC53, FAM21, SWIP, Strumpellin, and WASH (Derivery et al., 2009; Gomez and Billadeau, 2009; Jia et al., 2010). The SHRC is recruited to early endosomes by multivalent interactions between repeat elements in FAM21 and the Retromer subunit VPS35 (Harbour et al., 2012; Jia et al., 2012). WASH is essential for Arp2/3-induced F-actin accumulation on endosomes (Derivery et al., 2009; Gomez et al., 2012). With WASH suppression, scission of membrane tubules emanating from endosomes was suggested to be impaired (Derivery et al., 2009; Gomez and Billadeau, 2009) and knockout of WASH in MEFs resulted in the collapse of the early endosomal and lysosomal networks (Gomez et al., 2012).
Nucleation of F-actin by WASP family members is regulated by a variety of signaling events that converge on VCA motif activation/exposure (Padrick and Rosen, 2010). The VCA motifs of WASP/N-WASP, WAVE and WASH are autoinhibited through intramolecular and intermolecular interactions (Chen et al., 2010; Ismail et al., 2009; Jia et al., 2010; Kim et al., 2000; Miki et al., 1998). Several signaling molecules have been identified to promote the activation/exposure of WASP/N-WASP and WAVE VCA motifs to allow timed and localized F-actin nucleation, including small GTPases Cdc42 and Rac1, PIP2 and PIP3 phospholipids, and phosphorylation by the Src, Abl, and Cdk family of kinases (Eden et al., 2002; Ismail et al., 2009; Kim et al., 2000; Lebensohn and Kirschner, 2009; Miki et al., 1998; Padrick and Rosen, 2010). Contrary to WASP/N-WASP and WAVE, the mechanisms regulating WASH activation have been more elusive. The Rho GTPase has been genetically linked to activation of WASH in Drosophila (Liu et al., 2009), but is not sufficient to directly activate human WASH in vitro (Jia et al., 2010). Ubiquitination is a post-translational modification in which a small 76 amino acid protein is covalently attached to lysine residues in substrate proteins through a three step E1, E2, and E3 enzymatic cascade (Fang and Weissman, 2004).
Ubiquitination can have pleiotropic effects on its substrates depending on the length and type of ubiquitin chains. K48-linked ubiquitin chains typically target proteins for degradation by the 26S proteasome (Bochtler et al., 1999). However, K63-linked ubiquitination typically acts as a signaling event to modify function, such as altering protein-protein interactions or protein conformations, or targeting proteins for lysosomal delivery (Sun and Chen, 2004). Recently, we identified a novel class of proteins that bind to and enhance the activity of E3 RING ubiquitin ligases (Doyle et al., 2010). These ubiquitin ligase enhancers are known as melanoma antigen (MAGE) genes and comprise a family of over 50 unique human genes (Chomez et al., 2001). Although the biochemical function of MAGE proteins in ubiquitination has been elucidated, the cellular processes in which specific MAGE proteins act are unclear. Here we investigate the cellular function of MAGE-L2, a paternally imprinted gene that is abundantly expressed in the brain, maps to the Prader-Willi syndrome deletion locus, and is implicated in normal circadian rhythm and the hypothalamic-endocrine axis (Bischof et al., 2007; Boccaccio et al., 1999; Kozlov et al., 2007; Tennese and Wevrick, 2011).
To determine the specific E3 RING ubiquitin ligase partner of MAGE-L2 and gain insight into its cellular function, we examined MAGE-L2 binding partners by tandem affinity purification (TAP) coupled to mass spectrometry. The E3 RING ubiquitin ligase TRIM27 was identified as a major binding partner of MAGE-L2 (Figure 1A). Binding of MAGE-L2 and TRIM27 was confirmed by reciprocal co-immunoprecipitation experiments (Figures 1B and S1C). Moreover, in vitro translated MAGE-L2 bound recombinant GST-TRIM27 but not GST alone, indicating the interaction between the two proteins is direct (Figure 1C). To confirm the association of MAGE-L2 and TRIM27, we stably expressed GFP-MAGE-L2 and mCherry-TRIM27 in U2OS cells and examined their co-localization by live cell microscopy. We found that the two proteins co-localized in discrete cytoplasmic puncta (Figure 1D), as well as in a smaller nuclear pool (data not shown). These findings suggest that TRIM27 binds MAGE-L2 and that the MAGE-L2-TRIM27 ubiquitin ligase complex localizes to discrete cytoplasmic structures.
To determine the identity of the MAGE-L2-TRIM27 structures, we reanalyzed our mass spectrometry data of MAGE-L2 interacting proteins for clues. Interestingly, MAGE-L2 was identified to interact with VPS35 and VPS26 (Figure 1A), two components of the endosomal Retromer complex. mCherry-TRIM27 co-localized with GFP-tagged VPS35, VPS29, and VPS26, as well as the Retromer-associated SHRC proteins, WASH and FAM21 (Figure S1A). Furthermore, co-staining for endogenous VPS35 and TRIM27 clearly identified TRIM27 cytoplasmic structures as Retromer-positive endosomes (Figure 1E). Unfortunately, we were unable to observe endogenous MAGE-L2 due to the lack of specific, high quality antibodies (data not shown). More detailed analysis of TRIM27 localization revealed its localization to numerous tubular structures emanating from endosomes (Figure 1F), a property shared with the Retromer complex (Arighi et al., 2004; Bonifacino and Hurley, 2008). These findings suggest that MAGE-L2-TRIM27 localizes to the Retromer-positive subset of endosomes.
Next we confirmed the interaction between MAGE-L2-TRIM27 and the Retromer complex initially observed by mass spectrometry. MAGE-L2 and TRIM27 co-immunoprecipitated with all three components of the Retromer complex (Figures S1B–C and data not shown) and MAGE-L2 directly bound recombinant GST-VPS35 in vitro through its WH-B motif (Figures S1D and S1G–H). In addition, MAGE-L2 interaction with VPS35 did not impair VPS35 binding to the SHRC component FAM21 (Figure S1I). Furthermore, MAGE-L2-VPS35 interaction is functionally important, as knockdown of VPS35 dramatically inhibited MAGE-L2 and TRIM27 endosomal localization (Figures S1E–F and data not shown). These findings suggest that VPS35 recruits MAGE-L2-TRIM27 to Retromer-positive endosomes by binding to MAGE-L2.
We next assessed whether MAGE-L2-TRIM27 participates in endosome-to-Golgi transport. Two independent siRNAs targeting MAGE-L2 or TRIM27 were identified that significantly reduced their target protein’s levels, but had no effects on the levels of other known essential factors required for retrograde transport (Figure S2A–C). Multiple siRNAs targeting MAGE-L2 or TRIM27 resulted in impaired CI-M6PR and TGN46 trafficking to a degree similar to VPS35-RNAi (Figures 2A–C and S2D). Importantly, the overall organization of the TGN was unaffected in MAGE-L2- or TRIM27-RNAi cells (Figure S2E). The steady state defects in CI-M6PR localization were corroborated by examining transport of a small pool of surface localized CI-M6PR to the TGN in MAGE-L2- and TRIM27-RNAi cells. MAGE-L2- or TRIM27-RNAi impaired transport of surface-labeled CI-M6PR to the TGN (Figure 2D). Importantly, the dispersed steady state or surface labeled CI-M6PR in TRIM27- or MAGE-L2-RNAi cells co-localized with the endosomal marker EEA1 (Figures 2E and S2F). Furthermore, CI-M6PR protein levels were reduced in MAGE-L2- and TRIM27-RNAi cells and Cathepsin D processing and trafficking were impaired (Figure 2F). Finally, overexpression of MAGE-L2 in combination with TRIM27 increased CI-M6PR endosomal retrieval kinetics (Figure S2G). These results suggest that MAGE-L2-TRIM27 is required for endosome-to-Golgi retrograde transport.
To extend our findings, we examined the requirement of TRIM27 on the trafficking of two additional physiologically and pathologically relevant substrates of the Retromer and SHRC complexes. First, knockdown of TRIM27 significantly impaired trafficking of the Retromer cargo cholera toxin subunit B (CTxB) to the TGN (Figure 2G). However, the trafficking of the SHRC-independent cargo Transferrin receptor to perinuclear recycling endosomes was unaltered in TRIM27-RNAi cells (Figure 2G) (Duleh and Welch, 2010; Gomez and Billadeau, 2009; Gomez et al., 2012). In addition, the Retromer and SHRC complexes have been implicated in the recycling of endosomal proteins to the plasma membrane, including integrins (Duleh and Welch, 2012; Zech et al., 2011). Therefore, we examined whether MAGE-L2 and TRIM27 function extends beyond endosome-to-Golgi trafficking by examining their contribution to the recycling of integrin α5 to the plasma membrane. Knockdown of MAGE-L2 or TRIM27 significantly decreased cell surface integrin α5 (Figure 2H) and increased the intracellular pool of integrin α5 in EEA1-negative endosomes (Figure 2I). Importantly, this decrease in cell surface integrin α5 in MAGE-L2- or TRIM27-RNAi cells is functionally significant, as invasion of cells through matrigel, a process regulated by integrins, is significantly impaired in MAGE-L2- or TRIM27-RNAi cells (Figure 2J). These results suggest that MAGE-L2-TRIM27 functions with the Retromer and SHRC complexes in both endosome-to-Golgi and endosome-to-plasma membrane protein recycling.
Next we determined whether the requirement for MAGE-L2-TRIM27 in retrograde transport involved its ubiquitin ligase activity by utilizing a TRIM27 RING mutant lacking E3 ubiquitin ligase activity. Unlike wild-type TRIM27, expression of an RNAi resistant TRIM27 RING mutant was unable to rescue retrograde transport of CI-M6PR in TRIM27-RNAi cells (Figures 3A and S3A). Importantly, the TRIM27 RING mutant localized similarly to the wild-type protein (Figure S3B). These results suggest that the ubiquitin ligase activity of MAGE-L2-TRIM27 is important for proper retrograde transport.
Next we examined which of the more than 35 different E2 ubiquitin-conjugating enzymes functions with MAGE-L2-TRIM27 to facilitate retrograde endosome-to-Golgi trafficking of CI-M6PR. Of the 35 E2 enzymes examined, knockdown of only Ube2O and Ube2L6 resulted in dramatic alteration of CI-M6PR localization (Figures 3B, S3C, and data not shown). Ube2O-RNAi showed similar penetrance as TRIM27-RNAi (Figures 3B–C), resulted in CI-M6PR dispersion to EEA1-positive endosomes (Figure 3D), and reduced total CI-M6PR (Figure 3E). However, Ube2L6-RNAi did not redistribute CI-M6PR to EEA-positive endosomes (Figure 3D) or promote lysosomal degradation of CI-M6PR (Figure 3E), but rather disrupted TGN organization (Figure S3D). Notably, Ube2O, but not Ube2L6, was also identified in a yeast-2-hybrid screen for E2 enzymes interacting with TRIM27 (Markson et al., 2009). These results suggest that the Ube2O E2 ubiquitin-conjugating enzyme is the physiologically relevant E2 enzyme functioning with MAGE-L2-TRIM27 in supporting retrograde transport.
We next investigated whether retrograde transport was dependent on ubiquitin, and if so, which type of poly-ubiquitin chain. To do so, we utilized the previously developed system (Xu et al., 2009) to inducibly knockdown ubiquitin by addition of tetracycline and replace it with a ubiquitin variant that is unable to produce a specific ubiquitin chain, namely ubiquitin in which lysine 48 or lysine 63 has been mutated to arginine (K48R or K63R, respectively; Figure S3E–F). We found that depletion of ubiquitin from cells dramatically affected CI-M6PR trafficking (Figures 3F and S3G). Addition of wild-type or K48R ubiquitin completely rescued CI-M6PR localization (Figures 3F and S3G), suggesting that ubiquitin is required for retrograde trafficking, but K48-linked ubiquitin chains are not. In contrast, depletion of K63-ubiquitin chains dramatically blocked CI-M6PR TGN localization and relocalized it to EEA1-positive endosomes (Figures 3F and S3G and data not shown), but had no significant effect on the overall organization of the TGN (Figure S3H). Similarly to the steady state behavior, retrograde transport of cell surface labeled CI-M6PR to the TGN is also dependent on K63-linked ubiquitination (Figures 3G and S3I). Furthermore, depletion of K63-linked ubiquitin chains altered trafficking of the lysosomal hydrolase Cathepsin D, resulting in the reduction of mature Cathepsin D, accumulation of pro- and intermediate-Cathepsin D, and the secretion of pro-Cathepsin D into the cell culture media (Figure 3H). These results suggest that K63-linked ubiquitination is important for proper retrograde transport.
Next we assessed the precise mechanism by which MAGE-L2-TRIM27, Ube2O, and K63-linked ubiquitination promotes retrograde transport. Retrograde transport requires several ordered steps to facilitate endosome-to-Golgi transport, including endosomal localization of the Retromer complex and localization and activation of the SHRC to facilitate endosomal F-Actin accumulation by the Arp2/3 complex (Cullen and Korswagen, 2012). We interrogated several of these specific steps to determine when ubiquitination may be important. The localization of the Retromer complex to CI-M6PR substrate containing endosomes was unaffected by depletion of TRIM27 or K63-linked ubiquitin chains (Figure S4A–B). In addition, the SHRC was still recruited normally to Retromer-positive endosomes in TRIM27-RNAi cells (Figure S4C). However, knockdown of MAGE-L2 or TRIM27 resulted in reduced endosomal F-Actin to a degree similar to WASH-RNAi (Figure 4A–B). Similarly, knockdown of the physiologically relevant Ube2O E2 enzyme, but not Ube2L6, resulted in a reduced endosomal F-actin (Figure 4A–B). Consistent with reduced endosomal F-actin, knockdown of MAGE-L2, TRIM27, or Ube2O, but not Ube2L6, significantly reduced the accumulation of the Arp2/3 complex subunit, ARPC5, on SHRC-positive endosomes (Figure 4C–D). Notably, there was no defect in total ARPC5 or F-actin levels (Figure 4D and data not shown). Likewise, depletion of K63-linked ubiquitin chains resulted in the specific reduction of endosomal F-actin and Arp2/3 complex, without affecting total F-actin, Arp2/3 complex, or SHRC levels (Figures 4E–4H, S3J, and data not shown). Consistent with the previously described role of SHRC and endosomal F-actin in membrane tubule scission (Derivery et al., 2009; Gomez and Billadeau, 2009), TRIM27-RNAi, shUb-Ub, and shUb-Ub(K63R) cells accumulated endosomal tubules (Figure S4D–E). These results suggest that K63-linked ubiquitination by MAGE-L2-TRIM27 and Ube2O is required for localization of the Arp2/3 complex to the SHRC and generation of endosomal F-Actin.
We next examined whether the defective endosome-to-Golgi retrograde trafficking in TRIM27-RNAi or shUb-Ub(K63R) cells is due to decreased endosomal F-actin. First, we designed a fusion protein in which endosomal F-actin accumulation could be induced by an uninhibited WASH VCA motif. WASH VCA motif was fused to the C-terminal domain of FAM21 (Δ356N), which binds VPS35 for endosomal targeting (Figure 5A) (Gomez and Billadeau, 2009; Jia et al., 2012). Upon expression in cells, this fusion protein localized to Retromer-positive endosomes (Figure 5A) and increased F-Actin accumulation on endosomes (Figure 5A–B). We next determined whether this FAM21-WASH-VCA fusion could rescue retrograde transport in TRIM27-RNAi cells. Indeed this was the case for both Cathepsin D trafficking (Figure 5C) and CI-M6PR TGN localization (Figures 5D and S4F). Furthermore, the FAM21-WASH-VCA fusion rescued CI-M6PR trafficking in cells deficient for K63-linked ubiquitin chains (Figures 5E and S4G). Therefore, the primary requirement for TRIM27 and K63-linked ubiquitin chains in retrograde trafficking appears to be for accumulation of endosomal F-actin.
Generation of endosomal F-Actin is facilitated by the SHRC, which binds the Arp2/3 complex and actin through a conserved VCA domain in WASH (Derivery et al., 2009; Gomez and Billadeau, 2009). However, the WASH VCA domain exists in an autoinhibited state that must be relieved before nucleation of F-actin by the Arp2/3 complex can be achieved (Jia et al., 2010). Therefore, we speculated that MAGE-L2-TRIM27 may facilitate WASH-VCA exposure, Arp2/3 complex and actin binding, and consequent F-Actin nucleation on endosomes through targeted ubiquitination of the SHRC. Based on the highly homologous WAVE regulatory complex, regulators of SHRC activation are predicted to act on WASH itself or SWIP (Chen et al., 2010; Jia et al., 2010). Although we were unable to detect any ubiquitination of endogenous SWIP (Figure S5A), endogenous WASH was highly ubiquitinated (Figure 6A). WASH ubiquitination was TRIM27-dependent, as knockdown of TRIM27 dramatically reduced endogenous WASH ubiquitination (Figure 6B). Furthermore, WASH ubiquitination was K63-linked, as WASH was unable to be polyubiquitinated by K63R ubiquitin (Figure 6C). These findings suggest that TRIM27 is required for K63-linked ubiquitination of WASH.
Next we investigated the importance of WASH ubiquitination. To do so, we first determined the specific lysine in WASH that is conjugated to K63-linked ubiquitin chains. A single lysine, K220, in WASH had been identified in global proteomics studies to be ubiquitinated (Kim et al., 2011). Significantly, K220 is a highly conserved residue in species that express TRIM27 orthologues (Figure S5D). This residue is located in a region of WASH that is analogous to the “meander” region of WAVE, which is known to make contacts necessary for intra-complex inhibition in the WAVE regulatory complex (Chen et al., 2010). Therefore, we examined whether WASH ubiquitination was dependent on K220. Endogenous WASH was knocked down by RNAi and YFP-tagged wild-type or K220R WASH was re-expressed to insure integration of the re-expressed WASH into the SHRC. Unlike YFP-WASH wild-type, YFP-WASH K220R failed to be ubiquitinated (Figure 6C), suggesting that WASH ubiquitination is dependent on K220. We next examined whether WASH ubiquitination is important for retrograde transport. Using our re-expression system, YFP-WASH K220R, unlike wild-type YFP-WASH, could not support proper retrograde transport resulting in CI-M6PR dispersion (Figures 6D and S5B) and degradation (Figures 6E and S5B) and Cathepsin D secretion (Figure 6F). These results suggest that K63-linked ubiquitination of WASH K220 by TRIM27 is required for WASH function in retrograde transport.
We next examined whether WASH K220 ubiquitination may act as a signal to relieve WASH autoinhibition and promote its activity towards the Arp2/3 complex. As previously reported (Derivery et al., 2009), knockdown of WASH resulted in reduced endosomal Arp2/3 complex localization (Figures 6G and S5C). Unlike wild-type YFP-WASH, re-expression of YFP-WASH K220R was unable to rescue proper endosomal Arp2/3 complex localization (Figures 6G and S5C). Importantly, YFP-WASH K220R localized properly to endosomes indicating that it still incorporated into the SHRC (Figure S5C). These results suggest that ubiquitination of WASH K220 is required for WASH activation and Arp2/3 complex endosomal localization.
Finally, we developed an in vitro reconstitution system to directly test whether ubiquitination of WASH is required for its actin assembling activity. To do so, we reconstituted WASH knockout MEFs (Gomez et al., 2012) with stable expression of HA-GFP-WASH wild-type, ΔVCA, or K220R. The intact SHRC was then purified from each of these cell lines by anti-HA chromatography to near homogeneity (Figure S6A–B). Under these conditions, very little free WASH is present (Figure S6B). The activity of the reconstituted SHRCs was first assayed by examining their capacity to assemble F-actin on beads in cell lysates (Cory et al., 2003). As expected, SHRC stimulated F-actin accumulation on beads in a manner dependent on the VCA motif of WASH and was inhibited by cytochalasin D (Figure 7A). Importantly, WASH knockout MEFs reconstituted with WASH K220R mutant did not support actin assembly on beads (Figure 7A). In addition, the activity of SHRC was dependent on MAGE-L2-TRIM27, as SHRC isolated from MAGE-L2- or TRIM27-RNAi cells was significantly less active (Figure 7B). Furthermore, we examined the activity of purified SHRC in Arp2/3 complex-dependent pyrene-actin assembly assays. SHRC reconstituted with wild-type, ubiquitinated WASH displayed increased activity toward the Arp2/3 complex compared to SHRC reconstituted with non-ubiquitinated WASH K220R (Figure 7C). To further examine the interplay of WASH ubiquitination and activity, we treated purified wild-type SHRC with the K63-specific deubiquitinating enzyme AMSH. This treatment deconjugated K63-ubiquitin chains from WASH (Figure 7D) and significantly inhibited SHRC activity on beads (Figure 7E) and in pyrene-actin assembly assays (Figure 7F). These results suggest that K63-ubiquitination of WASH K220 by MAGE-L2-TRIM27 facilitates the activation of SHRC in a reversible manner.
To determine if ubiquitination of WASH K220 may facilitate SHRC activation by disrupting autoinhibitory contacts in the meander region around WASH K220, we mutated WASH K220 to aspartic acid (K220D) to destabilize autoinhibitory contacts in this region. Unlike inactive WASH K220R, WASH K220D is active in vitro (Figure 7A and 7C) and facilitates endosome-to-Golgi retrograde transport (Figure 6D–F) and endosomal Arp2/3 complex localization (Figure 6G) in cells. These results suggest that destabilizing the WASH meander region around the ubiquitination site can facilitate SHRC activity independent of ubiquitination.
MAGE proteins are a family of proteins that contain a conserved domain known as the MAGE homology domain. Recently, we showed that MAGE proteins function biochemically to bind to and enhance the activity of E3 RING ubiquitin ligases (Doyle et al., 2010). In this study we investigated the cellular function of one specific MAGE protein, MAGE-L2. Proteomic analysis revealed that MAGE-L2 specifically bound the TRIM27 E3 RING ubiquitin ligase. TRIM27 belongs to a large family of E3 RING ubiquitin ligases known as tripartite motif (TRIM) proteins. TRIM27 was originally identified and named Ret finger protein (RFP), due to its discovery as a gene that undergoes a translocation event with the Ret tyrosine kinase receptor in thyroid carcinomas (Saenko et al., 2003; Takahashi and Cooper, 1987). Subsequent work has implicated it in several processes, including transcriptional regulation, NFκB signaling, CD4+ T-cell homeostasis, and as an oncogene (Cai et al., 2011; Krutzfeldt et al., 2005; Shimono et al., 2000; Zha et al., 2006; Zoumpoulidou et al., 2012). It will be of particular interest in the future to determine if any of these diverse functions of TRIM27 are attributed to its regulation of WASH and vesicular transport.
Cellular studies revealed that MAGE-L2 and TRIM27 colocalized on cytoplasmic structures that were determined to be Retromer-positive endosomes. Of note, TRIM27 was previously shown to localize to similar structures that were unidentified at the time (Harbers et al., 2001). Furthermore, the localization of TRIM27 was regulated by PKC, JNK and RAS signaling pathways. To determine if any of these signaling pathways may contribute to the upstream regulation of MAGE-L2-TRIM27 and endosome-to-Golgi retrograde transport, we examined the effects of short-term inhibition of the PKC, JNK, MEK, and PI3K signaling pathways on the kinetics of surface CI-M6PR trafficking to the TGN. Specific inhibition of the JNK signaling pathway blocked CI-M6PR retrieval to the TGN (Figure S7). Future studies into the mechanism by which JNK signaling regulates endosomal protein recycling will be of particular interest.
Our mechanistic studies uncovered that K63-linked ubiquitination of WASH K220 by MAGE-L2-TRIM27 is required for endosomal F-actin nucleation and retrograde transport. WASH K220 is predicted to exist in an analogous region on the SHRC as the meander region on the known WAVE regulatory complex structure. The meander region makes contacts with WAVE itself and SRA1 (analogous to SWIP in SHRC) and is essential for maintaining WAVE in an inactive state (Chen et al., 2010). Importantly, this region is subjected to regulation by phosphorylation (Padrick and Rosen, 2010). Thus, the proposed meander region on both the WAVE and WASH regulatory complexes may be regulated by post-translational modifications, phosphorylation and ubiquitination, respectively. In addition, WASH K220 is highly conserved in vertebrates where TRIM27 is found. However, it is not conserved in invertebrates where TRIM27 orthologs are not present (Boudinot et al., 2011). Thus, regulation of SHRC by ubiquitination is likely highly conserved in vertebrates, but additional forms of regulation are predicted in invertebrates.
Our results suggest that ubiquitination of WASH facilitates activation by directly destabilizing autoinhibitory contacts in the SHRC, thus allowing VCA exposure, Arp2/3 complex binding, and subsequent F-actin assembly (Figure 7G). Consistently, destabilizing these autoinhibitory contacts bypasses the requirement for WASH ubiquitination. In the context of the cell, other factors likely cooperate to further enhance activity, perhaps by clustering the active SHRC. Another likely point of regulation is in deactivating WASH after sufficient endosomal F-actin nucleation for retrograde trafficking. Indeed, we find that ubiquitin-mediated activation of SHRC is reversible. Future studies into relevant deubiquitinating enzymes will be of interest.
Our findings also provide important insights into pathological conditions associated with MAGE-L2, TRIM27, and retrograde transport. Genes required for retrograde transport are frequently amplified in melanomas, contribute to tumorigenesis, and mediate trafficking of proteins required for tumor progression (Scott et al., 2009; Zech et al., 2011). Our findings extend this work to show that the TRIM27 oncogene facilitates integrin α5 recycling, an important event in tumorigenesis. Additionally, retrograde transport has been implicated in Alzheimer’s disease, where components of the pathway are downregulated and mutated (Small, 2008). Similarly, MAGE-L2 has been reported to be downregulated in the hippocampus of patients with incipient Alzheimer’s disease (Blalock et al., 2004). Finally, retrograde transport is an essential pathway in which many microbial toxins and some viruses enter cells. Indeed, inhibition of TRIM27 blocks trafficking of cholera toxin. Our results suggest a novel potential strategy to combat these pathogens.
Cells were cultured under standard conditions and transfected according to manufacturer’s recommendation. Detailed descriptions of cell culture conditions, transfection procedures, siRNA sequences, and antibodies are described in the Extended Experimental Procedures.
Tandem affinity purification (TAP) was performed using 293/TAP-Vector or 293/TAP-MAGE-L2 stable cell lines as described previously (Doyle et al., 2010) and in the Extended Experimental Procedures.
Immunoprecipitation and immunoblotting were performed as described previously (Potts and Yu, 2005). Cathepsin D secretion assay details are described in the Extended Experimental Procedures.
Recombinant proteins were produced using standard procedures described in the Extended Experimental Procedures. In vitro binding assays were performed as described previously (Doyle et al., 2010) and specified in the Extended Experimental Procedures.
Immunofluorescence was performed essentially as described previously (Potts and Yu, 2007) and in Extended Experimental Procedures. Retrograde transport of cell surface CI-M6PR was performed as described previously (Gomez and Billadeau, 2009) and detailed in the Extended Experimental Procedures.
WASH knockout MEFs (Gomez et al., 2012) were reconstituted with HA-GFP-tagged WASH and the resulting SHRC variants were purified as described in the Extended Experimental Procedures. Bead based and pyrene-actin assembly assays were performed as described previously (Cory et al., 2003; Jia et al., 2010) and detailed in the Extended Experimental Procedures.
We thank Mitsuaki Tabuchi (Kawasaki Medical School, Japan) for providing the Retromer construct. We also thank Potts lab members for helpful discussions and critical reading of the manuscript. This work was supported by the Sara and Frank McKnight Fellowship (PRP), Michael L. Rosenberg Scholar in Medical Research fund (PRP), Cancer Prevention and Research Initiative of Texas R1117 (PRP), the Mayo Foundation and National Institutes of Health Grant R01-AI065474 (DDB).
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