The E3 ubiquitin ligases RNF126 and Rabring7 regulate endosomal ...

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Research Article

The E3 ubiquitin ligases RNF126 and Rabring7 regulate endosomal sorting of the epidermal growth factor receptor Christopher J. Smith1,2, Donna M. Berry2 and C. Jane McGlade1,2,* 1

Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, ON, M5G 2M9, Canada The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, 101 College Street, Toronto, ON, M5G 1L7, Canada

2

*Author for correspondence ([email protected])

Journal of Cell Science

Accepted 2 January 2013 Journal of Cell Science 126, 1366–1380 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.116129

Summary Activation of the epidermal growth factor receptor (EGFR) results in internalization and ubiquitin-dependent endosomal sorting, leading to lysosomal degradation. Here we describe the role of the RING-finger-domain-containing protein RNF126 and the related protein, Rabring7 in EGFR endosomal sorting. We demonstrate that RNF126 specifies K48-linked chains with UbcH5b and also functions with Ubc13/Uev1a to form K63-linked chains in vitro. RNF126 and Rabring7 associate with the EGFR through a ubiquitin-binding zinc finger domain and both E3 ubiquitin ligases promote ubiquitylation of EGFR. In the absence of c-Cbl or in cells expressing Cbl-70Z, the binding of RNF126 and Rabring7 to the EGFR is reduced, suggesting that RNF126 and Rabring7 function downstream of c-Cbl. In HeLa cells depleted of either RNF126 or Rabring7 the EGFR is retained in a late endocytic compartment and is inefficiently degraded. In addition, depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. Together these data suggest that RNF126 and Rabring7 play a role in the ubiquitin-dependent sorting and downregulation of membrane receptors. Key words: Epidermal growth factor receptor, Multi-vesicular endosome, E3 ubiquitin ligase, K63 ubiquitin, RNF126, Rabring7

Introduction After binding to ligand the epidermal growth factor receptor (EGFR) is activated and removed from the cell surface. EGFR containing vesicles fuse with early endosomes, organelles that consist of both tubules and vacuoles, and which are Rab5 and transferrin receptor positive. From the early endosome, the EGFR can either be rapidly recycled back to the plasma membrane, or sorted towards the lysosome for degradation. EGFR containing early endosomes undergo a maturation process in preparation for fusion with the lysosome that is characterized by a loss of Rab5 and the acquisition of Rab7, a drop in intraluminal pH, the acquisition of lysosomal hydrolases as well as changes in endosome morphology. As opposed to early endosomes and recycling compartments, late endosomes are less tubulated and also harbor luminal vesicles (Huotari and Helenius, 2011). Once packaged into intra-luminal vesicles the EGFR is segregated from the cytosolic environment and can no longer access downstream signaling molecules. Therefore the process of endosome maturation is simultaneous with the downregulation of EGFinduced signaling cascades (Sorkin and Goh, 2008). The E3 ubiquitin ligase c-Cbl has emerged as a key regulator of EGFR downregulation. Upon ligand stimulation, c-Cbl associates with tyrosine phosphorylated EGFR and recruits the E2 Ubc4/5 leading to receptor ubiquitylation (Levkowitz et al., 1999; Umebayashi et al., 2008). There is mounting evidence to suggest that EGFR ubiquitylation is not required for receptor endocytosis but rather for the subsequent trafficking of the receptor towards the

lysosome for degradation. The EGFR is predominantly ubiquitylated on six lysines within the kinase domain and mutation of these residues to arginine does not reduce the rate of EGFR internalization but strongly inhibits EGFR degradation (Huang et al., 2006). Moreover, mutation of the direct binding site for c-Cbl on the EGFR (Y1045) did not affect EGFR internalization but impaired EGFR degradation (Grøvdal et al., 2004). Post-endocytic ubiquitin-dependent membrane traffic is mediated by the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins. ESCRT is composed of four protein complexes ESCRT-0, -I, -II and -III of which ESCRT-0, -I and -II contain proteins with ubiquitin binding domains. The ESCRT complexes concentrate ubiquitylated cargo on the limiting membrane of the endosome, drive inward budding, and catalyze membrane scission to form a multi-vesicular body (Henne et al., 2011; Hurley, 2010). Ubiquitin not only couples EGFR to the ESCRT machinery, recent studies have shown that EGF stimulation and receptor ubiquitylation stimulate the formation of multivesicular bodies (Eden et al., 2012; White et al., 2006). Here we characterize two E3 ubiquitin ligases termed RNF126 and Rabring7. We show that RNF126 can mediate the formation of K63-linked ubiquitin chains in vitro and that both RNF126 and Rabring7 associate with the EGFR through a ubiquitin-binding zinc finger. Both RNF126 and Rabring7 ubiquitylate the EGFR and in cells depleted of either RN126 or Rabring7 the EGFR is retained in a late endocytic compartment. In addition, we show that the depletion of Rabring7 attenuates the degradation of MET

RNF126 and Rabring7 regulate endosomal sorting of EGFR and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. Lastly, we show that the knockdown of RNF126 or Rabring7 alters the sorting machinery found at the late endosome and inhibits the formation of multivesicular bodies. Taken together, our data suggest that RNF126 and Rabring7 regulate the ubiquitin-dependent sorting of cell surface receptors through the endocytic system. Results


RNF126 mediates the formation of K48-linked chains with UbcH5b, and K63-linked chains with Ubc13/Uev1a

RNF126 and Rabring7 are related proteins that both contain a Cterminal RING finger domain and N-terminal zinc finger domain (Fig. 1A). Rabring7 has previously been shown to have RINGfinger-dependent auto-ubiquitylation activity (Amemiya et al., 2008; Burger et al., 2005; Sakane et al., 2007). To determine whether RNF126 exhibits RING-finger-dependent E3 ligase activity, we used in vitro auto-ubiquitylation assays with the E2 enzyme UbcH5b. GST–RNF126 and GST–Rabring7 as well as the isolated RING finger domain of each protein mediated the formation of polyubiquitin chains in these reactions. To disrupt the E2 interaction with the RING finger domain of RNF126 or Rabring7, we changed a zinc chelating cysteine within this domain to an alanine. Both GST–RNF126C231A and GST– Rabring7C229A failed to support ubiquitylation with UbcH5b indicating that the RING finger domain of RNF126 and Rabring7 are necessary to support polyubiquitylation in conjunction with UbcH5b (Fig. 1B, left panels). The identity of the E2 enzyme specifies the lysine of the acceptor ubiquitin that will be used to build a poly-ubiquitin chain (Ye and Rape, 2009). The formation of ubiquitin chain linkages at lysine 63 are often driven by the E2 Ubc13 which heterodimerizes with the Uev E2 variants (Hofmann and Pickart, 1999). Based on the sequence similarity between the RING finger domain of RNF126 to other RING finger domains predicted to contact Ubc13 (Plans et al., 2006), we investigated whether RNF126 and Rabring7 mediate the formation of K63 linked chains with Ubc13/Uev1a. When included in the in vitro ubiquitylation assays with Ubc13/Uev1a, only RNF126 mediated the formation of polyubiquitin chains. Furthermore, the isolated RING finger was not sufficient for ubiquitylation in the presence of Ubc13/Uev1a suggesting that a region outside the RING finger of RNF126 may be required to contact Ubc13/Uev1a (Fig. 1B, right panels). In addition to a C-terminal RING finger domain, an N-terminal zinc finger domain in both RNF126 and Rabring7 has been predicted (Amemiya et al., 2008; Burger et al., 2006). To disrupt this domain, we employed site directed mutagenesis to substitute two cysteine residues that are predicted to chelate a zinc ion to alanines. When RNF126C13,16A as well as Rabring7C22,25A were included in the reactions, ubiquitin chains were still formed with equal or greater efficiency indicating that the zinc finger is not required for in vitro ubiquitylation (Fig. 1B). To examine the ubiquitin chain topology formed by RNF126 or Rabring7 with either UbcH5b or Ubc13/Uev1a, we included ubiquitin mutants where lys63 or lys48 were replaced with arginine residues in the reactions. In the presence of UbcH5b, RNF126 formed ubiquitin chains with wild type ubiquitin and the K63R ubiquitin mutant while no ubiquitylation was observed when the K48R ubiquitin mutant was included in the reactions indicating that RNF126-UbcH5b selectively builds K48-linked chains. However, in the presence of the zinc finger mutant

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RNF126C13,16A, K48R ubiquitin was efficiently incorporated into the poly-ubiquitin chains suggesting that the zinc finger domain restricts the topology of ubiquitin chains formed by RNF126UbcH5b. In conjunction with Ubc13/Uev1a, both RNF126 and RNF126C13,16A assembled poly-ubiquitin chains with wild-type ubiquitin or the K48R but not with K63R ubiquitin confirming that RNF126 in conjunction with Ubc13/Uev1a builds K63linked chains. In the reactions containing UbcH5b and either Rabring7 or Rabring7C22,25A, wild-type (WT) ubiquitin, K48R ubiquitin and K63R ubiquitin were all efficiently incorporated into ubiquitin chains suggesting that the Rabring7-UbcH5b builds ubiquitin chains of mixed linkage (Fig. 1C). The zinc finger domain of RNF126 and Rabring7 bind directly to ubiquitin

Recent structural studies have defined a role for zinc finger domains as ubiquitin interaction modules and Rabring7 has been shown to associate with ubiquitin (Amemiya et al., 2008; Burger et al., 2006; Dikic et al., 2009). This lead us to further characterize the ability of RNF126 and Rabring7 zinc finger domains to bind to ubiquitin. GST fusions of RNF126 and Rabring7 were incubated with purified K48or K63-linked poly-ubiquitin chains (2–7 Ub in length). Full-length WT RNF126 and Rabring7 as well as the isolated zinc finger domain of each protein bound to both K48 and K63 chains of different lengths. When RNF126C13,16A as well as Rabring7C22,25A were mixed with the ubiquitin chains, no binding was observed confirming that the zinc finger domain of RNF126 and Rabring7 mediate direct binding to K48- and K63-linked polyubiquitin chains (Fig. 1D). Depletion of RNF126 or Rabring7 delays EGFR degradation and prolongs EGFR phosphorylation

Previous studies have shown that Rabring7 interacts with the Rab7 GTPase and there are conflicting reports as to whether the overexpression of Rabring7 attenuates or accelerates EGFR degradation (Mizuno et al., 2003; Sakane et al., 2007). To test the potential role of RNF126 and Rabring7 in EGFR trafficking, we used shRNA to create clonal HeLa cell lines stably depleted of either RNF126 or Rabring7 (Fig. 2A) and examined EGFR degradation after ligand stimulation by western blot. In both the RNF126- and Rabring7-knockdown lines, an increase in EGFR levels was observed at the 30 min time point suggesting that the depletion of RNF126 or Rabring7 delays the degradation of the EGFR after EGF stimulation (Fig. 2B). A similar delay in EGFR degradation was observed in HeLa cells stably depleted of Rabring7 using an shRNA construct with a different sense strand sequence as well as HeLa cells treated with siRNA directed against a different region of RNF126 than the sense sequence of the shRNA (supplementary material Fig. S2). To test whether the co-depletion of RNF126 and Rabring7 would further attenuate EGFR degradation, we treated the Rabring7-knockdown lines with siRNA directed against RNF126 and again monitored EGFR degradation by western blot. The depletion of both RNF126 and Rabring7 had no additional effect on EGFR degradation compared to Rabring7 knockdown alone (Fig. 2C). We next investigated EGFR phosphorylation and the association of the EGFR with downstream tyrosine phosphorylated proteins. Anti-phosphotyrosine western blots of EGFR immunoprecipitates showed that EGFR phosphorylation was enhanced 30 to 45 min post-stimulation in both RNF126 and Rabring7 knockdown compared to the non-silencing control and exhibited an enhanced association with tyrosine-phosphorylated proteins including SHC (Fig. 2D).


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Journal of Cell Science 126 (6)

Fig. 1. RNF126 and Rabring7 are RING finger E3 ubiquitin ligases. (A) RNF126 and Rabring7 have a similar domain architecture. Schematic representation of RNF126 and Rabring7 showing the N-terminal zinc finger (ZnF) and C-terminal RING finger domain of each protein. The amino acid sequence similarity between regions of the two proteins is indicated. (B) RNF126 has RING-finger-dependent ubiquitin ligase activity with UbcH5b and Ubc13/Uev1a. Full-length GST fusion proteins were incubated with ATP, ubiquitin, E1 and the indicated E2 for 90 min at 30 ˚C. RNF126 and Rabring7 fusion proteins containing mutations in the RING domain (C231A) and (C229A), ZnF domain (C13, 16A) and (C22, 25A) and the isolated RING finger domains were also included. In the control lanes (-UB, -E2, -E3) the indicated component was excluded from the reaction. Auto-ubiquitylation activity was detected by antiubiquitin western blotting. (C) K48-linked ubiquitin chains are formed by RNF126–UbcH5b whereas K63-linked chains are formed by RNF126–Ubc13/Uev1a. Ubiquitylation reactions were carried out as in B with the exception that ubiquitin mutants containing a lysine to arginine substitution at position 48 or at position 63 were included in the reactions. (D) The zinc finger domains of RNF126 and Rabring7 are necessary and sufficient to mediate their association with K48 and K63 ubiquitin chains. Purified K48-linked or K63-linked ubiquitin chains of different lengths (ub2–ub7) were incubated with the indicated immobilized GST fusion proteins, and bound ubiquitin chains were detected by western blotting with anti-ubiquitin. The pattern of the ubiquitin chains included in the reaction are shown on the right.

RNF126 and Rabring7 regulate endosomal sorting of EGFR

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Fig. 2. Depletion of either RNF126 or Rabring7 delays ligand-induced EGFR degradation and prolongs EGFR phosphorylation. (A) HeLa cells depleted of either RNF126 or Rabring7 were generated. Cells expressing a non-silencing shRNA or an shRNA construct targeting RNF126 were lysed and immunoblotted with an anti-RNF126 antibody. Arrow indicates the band corresponding to the predicted size of RNF126 (left panel). HeLa cells expressing a non-silencing shRNA or an shRNA construct targeting Rabring7 were lysed and Rabring7 was immunoprecipitated and analyzed with an antibody against Rabring7. The arrow indicates the band corresponding to the predicted size of Rabring7 (right panel). (B) Depletion of RNF126 or Rabring7 delays EGFR degradation after ligand stimulation. HeLa cells expressing a nonsilencing shRNA or cells depleted of either Rabring7 or RNF126 were serum starved and stimulated with 20 ng/ml EGF for the indicated times before lysis in RIPA buffer. 20 mg of protein lysate was resolved by SDS-PAGE and immunoblotted with an antibody against EGFR or b-tubulin. The mean EGFR immunoreactivity from three biological replicates, relative to the 10 min time point is plotted. Error bars represent standard error of the mean. (C) The degradation of the EGFR is not further attenuated in cells depleted of both Rabring7 and RNF126. The Rabring7knockdown line was transfected with a negative control siRNA or an siRNA directed against RNF126. 48 hours posttransfection, cells were stimulated with EGF and lysed as described above. Lysate was analyzed by immunobotting with antibodies against EGFR, RNF126 and btubulin. (D) Knockdown of either RNF126 or Rabring7 prolongs EGFR phosphorylation and its association with tyrosine phosphorylated proteins. Nonsilencing, RNF126-knockdown as well as Rabring7-knockdown lines were serum starved and stimulated with 20 ng/ml EGF, and the EGFR was then immunoprecipitated and immunoblotted with antibodies against phosphotyrosine and SHC.

The depletion of RNF126 and Rabring7 attenuates the degradation of MET and CXCR4

To investigate whether RNF126 or Rabring7 play a role in the sorting of other receptors known to undergo ligand-induced ubiquitylation and lysosomal degradation also we examined downregulation of MET and CXCR4. MET RTK is ubiquitylated by c-Cbl after stimulation with hepatocyte growth factor (HGF) (Peschard et al.,

2001). Uncoupling c-Cbl from MET delays the degradation of the receptor suggesting that MET, like the EGFR, undergoes ubiquitindependent downregulation. Moreover, Met phosphorylates components of the ESCRT machinery suggesting that ubiquitylated MET is sorted by ESCRT (Abella et al., 2005). In cell lines depleted of RNF126 the kinetics of MET degradation after HGF stimulation was comparable to control. In contrast, MET degradation was

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attenuated in cells depleted of Rabring7 suggesting that Rabring7, but not RNF126, is involved in the sorting of MET (Fig. 3A). We also examined the degradation of the G protein coupled receptor CXCR4. CXCR4, chemokine receptor type 4, is ubiquitylated by the E3 AIP4 in response to stimulation with SDF-1a (Marchese et al., 2003).

Depletion of HRS (Marchese et al., 2003), STAM-1 (Malik and Marchese, 2010) or Vps22 (Malerod et al., 2007) alter CXCR4 degradation suggesting that sorting of this receptor is dependent on the ESCRT machinery. We transiently expressed HA–CXCR4 in both control and knockdown lines and determined the volume of

Fig. 3. RNF126 and Rabring7 in the regulation of MET and CXCR4 sorting. (A) The depletion of Rabring7 delays the degradation of MET. HeLa cells expressing a non-silencing shRNA or cells stably depleted of either RNF126 or Rabring7 were serum starved overnight, treated with 100 ng/ml cycloheximide for 2 hours and subsequently stimulated with 20 ng/ml HGF for the indicated times and lysed. Lysates were analyzed by immunoblotting with antibodies against MET and b-tubulin. The mean MET immunoreactivity from three different experiments relative to the levels before stimulation is plotted. Error bars show the standard error of the mean (s.e.m.); *P50.034, n53. (B) RNF126 and Rabring7 regulate the sorting of CXCR4 at the early endosome. Nonsilencing and knockdown lines were transfected with an equivalent amount of HACXCR4. 24 hours post-transfection, cells were serum starved for 5 hours in the presence of 100 ng/ml cycloheximide and stimulated for 3 hours with 240 ng/ml SDF-1a before being fixed and labeled with anti-HA (red) and antiEEA1 (green). Scale bars: 10 mm. The three rightmost images are enlargements of the boxed regions in the second column. (C) The depletion of RNF126 and Rabring7 delays the degradation of CXCR4. The volume of CXCR4 above a threshold defined by the least intense CXCR4 punctae was measured. Data are presented as the volume (mm3) of Cy3 over threshold (15,000 AU)/total cellular volume Cy3 (mm3) measured. Values are means 6 s.e.m. (D) CXCR4 is colocalized with EEA1 in cells depleted of RNF126 or Rabring7. Values are mean Mander’s coefficient 6 s.e.m.

RNF126 and Rabring7 regulate endosomal sorting of EGFR vesicular CXCR4 staining after treating the cells for 3 hours with SDF-1a (Fig. 3B). Compared to the control lines, both RNF126- and Rabring7-knockdown lines contained a greater volume of CXCR4 (Fig. 3C). In addition, a greater degree of colocalization was observed with EEA1, a marker of early endosomes in the knockdown lines compared to control (Fig. 3D). This data suggests that in addition to regulating the late lysosomal degradation of RTKs, RNF126 and Rabring7 also regulate the sorting of CXCR4 from EEA1-positive early endosomes.


RNF126 and Rabring7 associate with the EGFR downstream of c-Cbl

Given the role for ubiquitin in RTK downregulation, we examined whether RNF126 and Rabring7 interact with the EGFR. Lysates from HeLa cells that were stimulated with EGF for the indicated time points were mixed with GST–RNF126 or GST–Rabring7. GST–RNF126 and GST–Rabring7 only associated with the EGFR after ligand stimulation with the most EGFR being bound at the 5 min time point (Fig. 4A). To investigate whether the ubiquitin-binding zinc finger of RNF126 and Rabring7 mediated the association with the EGFR, fusion proteins bearing mutations in the zinc finger domains as well as the isolated zinc finger domains of each protein were incubated with lysate from HeLa cells that had been stimulated with ligand for 5 min. GST–RNF126 and GST–Rabring7 associated with EGFR which was phosphorylated and ubiquitylated. The ubiquitin-binding zinc finger domain is necessary for this interaction as GST–RNF126C13,16A and GST–Rabring7C21,25A failed to associate with the EGFR. No interaction between the isolated zinc finger domains and the EGFR was observed suggesting that the zinc finger domain of either RNF126 or Rabring7 is necessary but not sufficient to bind the EGFR (Fig. 4B). We also investigated whether RNF126 and Rabring7 associate with FLT3, a representative member of the class III RTKs. Ba/F3 cells that overexpress FLT3 were either stimulated with FLT3L for 5 min or left unstimulated and incubated with the indicated fusion proteins. Both GST–RNF126 and GST–Rabring7 associated with tyrosine phosphorylated FLT3 after ligand stimulation. While some ligand-independent binding to FLT3 was observed, GST–RNF126 and GST–Rabring7 binding to a high molecular weight FLT3 species predicted to represent phosphorylated and ubiquitylated receptor was dependent on FLT3 ligand stimulation. GST–RNF126C13,16A and GST– Rabring7C22,25A failed to associate with the high molecular weight FLT3 species while the isolated zinc finger domain in this system bound to a tyrosine phosphorylated protein corresponding to the predicted size of FLT3 (supplementary material Fig. S3). Given that the ubiquitin-binding zinc finger is required for the association of RNF126 and Rabring7 to the EGFR we sought to investigate whether RNF126 and Rabring7 binding to the receptor was contingent upon c-Cbl. We compared the association between the EGFR and RNF126 or Rabring7 in murine embryonic fibroblast (MEF) cells derived from either WT mice or c-Cbl2/2 mice (Duan et al., 2003). WT and c-Cbl2/2 MEFs were stimulated with EGF and the lysate was mixed with equivalent amounts of either GST–RNF126 or GST–Rabring7. GST–RNF126 and GST–Rabring7 associate with the EGFR in lysate derived from the WT MEFs stimulated with EGF for 2 and 10 min. In lysate derived from the c-Cbl2/2 MEFs we observed decreased association of GST–RNF126 and GST–RNF126 to the

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EGFR at both time points (Fig. 4C). We also compared the association of GST–RNF126 and GST–Rabring7 with the EGFR in lysate derived from HeLa cells transfected with an empty vector control or HeLa cells transiently transfected with HA-Cbl70Z, a dominant negative form of c-Cbl (Andoniou et al., 1994; Waterman et al., 1999). The expression of HA-Cbl-70Z reduced the binding of GST–RNF126 and GST–Rabring7 to the EGFR (Fig. 4D). Taken together, these results suggest that c-Cbl is required for the effective recruitment of RNF126 and Rabring7 and suggests that RNF126 and Rabring7 function downstream of c-Cbl. The EGFR is a substrate of RNF126 and Rabring7

To determine whether EGFR is a substrate for RNF126 mediated ubiquitylation. HEK-293T cells were co-transfected with EGFRFLAG, HA-UB and either empty vector, RNF126 or RNF126 C231A and subsequently serum starved overnight, stimulated with 100 ng/ml EGF and lysed in boiling RIPA buffer containing 1% SDS. EGFR ubiquitylation was enhanced in cells cotransfected with RNF126 compared to the vector control and cells transfected with RNF126 C231A (Fig. 5A). To investigate whether RNF126 and Rabring7 associate with and ubiquitylate EGFR directly, we examined RNF126 and Rabring7 mediated EGFR ubiquitylation in vitro. The cytoplasmic region of EGFR was transcribed and translated in a wheat germ extract system in the presence of [35S]methionine and subsequently included in ubiquitylation assays containing either GST–RNF126 or GST– Rabring7 in the presence of either UbcH5b or Ubc13/Uev1a as the E2 enzyme. The cytoplasmic domain of EGFR appeared as a high molecular weight smear in the reactions containing GST– RNF126 with UbcH5b or Ubc13/Uev1a indicative of modification with ubiquitin (Fig. 5B). EGFR was also significantly modified in the presence of GST–Rabring7 with UbcH5b (Fig. 5C). We performed a similar in vitro assay with the cytoplasmic region of FLT3. FLT3 appeared as a high molecular weight smear in the reactions containing GST– RNF126 or GST–Rabring7 with UbcH5b and in the reactions containing GST–RNF126 with Ubc13/Uev1a (supplementary material Fig. S4). To test whether the depletion of RNF126 or Rabring7 altered the ubiquitylation of the EGFR we used a previously described HEK-293 cell line expressing FLAG-tagged EGFR (HEK-EGFR) (Tong et al., 2008). We used shRNA to establish HEK-EGFR cell lines stably depleted of either RNF126 or Rabring7 (Fig. 5D). We stimulated control and knockdown cells with EGF and examined the pattern of ubiquitylation in FLAG immunoprecipitates. In the control lines, the EGFR is ubiquitylated at the 10 and 30 min time points post-EGF stimulation. In cells depleted of RNF126, receptor ubiquitylation is only observed after 30 min of ligand stimulation. In cells depleted of Rabring7, a reduction in EGFR ubiquitylation is observed at both 10 and 30 min time points (Fig. 5E). Together this data suggest that RNF126 and Rabring7 are able to directly ubiquitylate the EGFR and that the depletion of these E3 ubiquitin ligases alter the dynamics of EGFR ubiquitylation. The EGFR is retained in a late endocytic compartment in the absence of RNF126 or Rabring7

To investigate the progression of the EGFR through the endocytic pathway we examined the distribution of the EGFR at various time points post-stimulation. In the RNF126- and Rabring7-knockdown lines, we noted that the EGFR had a



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Fig. 4. The zinc finger domains of RNF126 and Rabring7 mediate their association with activated EGFR. (A) GST–RNF126 and GST– Rabring7 associate with EGFR in a ligand-dependent manner. Immobilized GST–RNF126 or GST–Rabring7 were incubated with cell lysate derived from HeLa cells that were serum starved and either left unstimulated or incubated with 20 ng/ml EGF for the indicated time period. Bound material was immunoblotted with antibodies against EGFR. (B) The zinc finger domain of RNF126 and Rabring7 is required for EGFR binding. GST fusions of full-length, ZnF mutant (C13, 16A) and (C22, 25A) or the isolated ZnF region were incubated with cell lysate from HeLa cells stimulated for 5 min with EGF. Bound material was analyzed by immunoblotting with anti-ubiquitin or blotted with anti-phosphotyrosine and anti-EGFR. (C) The association between RNF126 or Rabring7 with the EGFR is reduced in c-Cbl2/2 MEFs. Immobilized GST–RNF126 or GST–Rabring7 were incubated with cell lysate derived from c-Cbl+/+ or c-Cbl2/2 MEFs, which were serum starved for 16 hours and stimulated with 100 ng/ml EGF for the indicated time. Bound material was immunoblotted with antibodies against EGFR. (D) The expression of HA–Cbl-70Z reduces the association between RNF126 or Rabring7 and the EGFR. HeLa cells transfected with either an empty vector control or HA-Cbl-70Z were serum starved, stimulated for 5 min with 20 ng/ml EGF and lysed. Lysate was mixed with equivalent amounts of immobilized GST, GST– RNF126 or GST–Rabring7. Bound material was immunoblotted with antibodies against the EGFR.

tendency to remain in punctae at the 60 min time point compared to the control lines where the majority of the EGFR had been degraded (supplementary material Fig. S5). To quantify these observations, we compared the relative volume of EGFR staining above an intensity threshold that corresponded to the least intense EGFR vesicle. In the control lines, the relative volume of EGFR

above threshold peaked at 10 min post-EGF stimulation and decreased in subsequent time points. In the knockdown lines, the relative volume of EGFR above threshold continued to increase after the 10 min time point suggesting that in cells depleted of RNF126 or Rabring7, the EGFR accumulates in an endocytic compartment (Fig. 6A,B). To determine the identity of this

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Fig. 5. RNF126 and Rabring7 ubiquitylate the EGFR. (A) HEK-293T cells were co-transfected with HA-UB, EGFR-FLAG and either pcDNA3.1 empty vector, RNF126 or RNF126C231A. Cells were serum starved for 16 h after transfection and stimulated with 100 ng/ml EGF for 10 min as indicated. Cells were lysed in boiling RIPA buffer containing 1% SDS, passed through a syringe and diluted tenfold before immunoprecipitating FLAG-tagged EGFR. Immunoprecipitates were analyzed by immunoblotting with an anti-HA antibody and lysates were analyzed by immunoblotting with antiFLAG and anti-RNF126. (B) RNF126 ubiquitylates EGFR in vitro. The cytoplasmic region of the EGFR was transcribed and translated in a wheat germ extract system in the presence of [35S]methionine. Radiolabeled EGFR was added to an in vitro ubiquitylation reaction containing ATP, ubiquitin, E1 and either UbcH5b or Ubc13/Uev1a in the presence or absence of GST–RNF126 or GST–RNF126 C231A. Reactions were separated by SDS-PAGE and visualized by autoradiography. (C) Rabring7 ubiquitylates EGFR in vitro. In vitro translated EGFR was included in a ubiquitylation reaction containing ATP, ubiquitin, E1 and UbcH5b in the presence or absence of GST–Rabring7 or GST–Rabring7C229A. The arrow indicates the unmodified cytoplasmic region of EGFR. (D) The depletion of RNF126 or Rabring7 alters the pattern of ubiquitylation on the EGFR. HEK-EGFR cells stably expressing a nonsilencing shRNA or an shRNA construct directed against RNF126 or Rabring7 were generated. Cells were lysed and immunoblotted with an anti-RNF126 antibody. Rabring7 was also immunoprecipitated and analyzed with an antibody directed against Rabring7 (right panels). Non-silencing, RNF126knockdown (KD) or Rabring7-knockdown HEK-EGFR were serum starved and stimulated with 100 ng/ml EGF for the indicated times before lysis in RIPA buffer containing 1% SDS. They were then passed through a syringe and diluted tenfold before FLAG-tagged EGFR was immunoprecipitated. Immunoprecipitates were analyzed by immunoblotting with an anti-ubiquitin antibody. Lysate was analyzed by immunoblotting with anti-FLAG and b-tubulin antibodies.



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Fig. 6. See next page for legend.

RNF126 and Rabring7 regulate endosomal sorting of EGFR compartment, we co-stained with either the early endosomal marker EEA1 or CD63, a marker of late endocytic compartments. After 10 min EGF stimulation, the distribution of the EGFR with respect to EEA1 and CD63 was comparable between the nonsilencing lines and the knockdown lines with the majority of the EGFR in an EEA1-positive compartment. After 60 min of EGF stimulation, a time point at which little vesicular EGFR remains in the control line, EGFR was predominantly colocalized with CD63 in the knockdown lines (Fig. 6A,C). Together, this suggests that in cells depleted of RNF126 or Rabring7, the EGFR progresses normally through early endosomes but is inefficiently sorted at the late endosome towards the lysosome for degradation.


The depletion of RNF126 or Rabring7 alters the EGFR sorting machinery

Deregulated EGFR traffic has been attributed to becoming uncoupled from c-Cbl and its associated proteins (Dikic and Schmidt, 2007). For example, mutation of the direct binding site for c-Cbl on the EGFR (Y1045) leads to decreased receptor degradation and retention within early endosomes (Grøvdal et al., 2004). To determine if RNF126 or Rabring7 depletion affected cCbl binding to EGFR, we examined their association by immunoprecipitation. While the amount of EGFR in the c-Cbl IP was comparable between non-silencing and RNF126knockdown lines 10 min post-stimulation, c-Cbl and the EGFR continued to associate 30 min post-stimulation in the RNF126knockdown line (Fig. 7A). This suggests that in spite of the continued association of c-Cbl, the EGFR fails to be efficiently targeted to the lysosome in the absence of RNF126. To further our characterization of RNF126, we sought to identify other interacting proteins using mass spectrometry. We identified YMER as a potential RNF126 interacting protein and confirmed binding of myc-YMER to GST–RNF126 (Fig. 7B, left panel). YMER is an EGFR interacting protein that is thought to inhibit EGFR downregulation through an unknown mechanism. EGF stimulation induces the phosphorylation and ubiquitylation of YMER and YMER phosphorylation is thought to regulate its function in attenuating EGFR degradation (Tashiro et al., 2006). In the RNF126-knockdown line we found that the EGF-induced phosphorylation of YMER was decreased compared to the nonsilencing control (Fig. 7B, right panel) suggesting that YMER may form a complex with RNF126 that could be involved in modulating the downregulation of the EGFR. We also identified SCAMP3 as a potential RNF126 interacting protein and Fig. 6. The EGFR is retained in the late endocytic compartment in cells depleted of either RNF126 or Rabring7. (A) Non-silencing, RNF126- or Rabring7-knockdown lines were seeded on glass coverslips, serum starved and stimulated for up to 1 hour with 20 ng/ml EGF. Cells were subsequently fixed, permeabilized and incubated with an anti-EGFR antibody (red) together with either anti-EEA1 or anti-CD63 (green). The far right panels show a magnified view of the boxed regions in the previous panels. Scale bars: 10 mm. (B) The EGFR accumulates in intracellular punctae in cells depleted of RNF126 or Rabring7. The volume of EGFR above a threshold defined by the least intense EGFR punctae was measured. Data are presented as the volume (mm3) of Cy3 over threshold (5000 AU)/total cellular volume (mm3) measured. Values are means 6 s.e.m. (C) The EGFR is colocalized with CD63 in the knockdown lines 60 min after EGF stimulation. The mean Pearson’s correlation of EGFR with EEA1 or CD63 is shown. Values are means 6 s.e.m.

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confirmed binding of endogenous SCAMP3 to GST–RNF126 (Fig. 7D, left panel). SCAMP3 physically associates with Tsg101 and the depletion of SCAMP3 reduces the formation of multivesicular bodies after EGF stimulation. SCAMP3 plays a role in the post-endocytic downregulation of the EGFR and is tyrosine phosphorylated by the EGFR after EGF stimulation (Aoh et al., 2009; Falguieres et al., 2012; Wu and Castle, 1998). We found that the EGF-induced phosphorylation of SCAMP3 was decreased in the RNF126-knockdown line (Fig. 7D, right panel), suggesting that RNF126 and SCAMP3 may form a complex that could be involved in sorting the EGFR. We investigated whether RNF126 like Rabring7 associates with the GTPase Rab7. Rab7 localizes to the late endosome, is a key regulator of the endosome to lysosome fusion and also plays a role in EGFR downregulation (Bucci et al., 2000; Vanlandingham and Ceresa, 2009). When cotransfected in COS cells, HA–RNF126 and a truncation of HA– RNF126 lacking the N-terminal zinc finger domain coimmunoprecipitated with GFP–Rab7 (supplementary material Fig. S6A). To date we have not been able to establish conditions to reliably detect the localization of endogenous RNF126 with respect to the Rab GTPases. RNF126 and Rabring7 binding to Rab7 raised the possibility that the retention of the EGFR in the late endosome in the RNF126-knockdown line was due to a deregulation of Rab7 function. However, the distribution and intensity of endogenous Rab7 was comparable between control and both RNF126- and Rabring7-knockdown lines suggesting that the depletion of RNF126 or Rabring7 does not affect Rab7 (supplementary material Fig. S6B). To further investigate endosome maturation we quantified the volume of acidic compartments in control and RNF126 or Rabring7 depleted cells using LysoTracker, an acidotropic dye. The volume of LysoTracker staining was comparable between control and knockdown lines suggesting that the depletion of RNF126 or Rabring7 does not affect the acidification of endosomes (supplementary material Fig. S6C). In addition, the distribution and intensity of CD63 and lysobisphosphatidic acid (LBPA), a lipid found within the lumen of multivesicular bodies, was comparable between control and knockdown lines suggesting that the depletion of RNF126 or Rabring7 does not alter late endosome motility (supplementary material Fig. S6D). We next sought to investigate whether the depletion of RNF126 or Rabring7 had effects on the ESCRT machinery. While the total levels of HRS, TSG101 and Vps24 which belong to the ESCRT-0, ESCRT-I and ESCRT-III complexes respectively were comparable between non-silencing and knockdown lines, the levels of Vps22 a component of ESCRT-II was reduced in both RNF126 and Rabring7 knockdown lines (Fig. 7C). Consistent with our results, the depletion of Vps22 has previously been shown to attenuate both EGFR and CXCR4 degradation (Malerod et al., 2007). Taken together, this suggests that the endocytic compartments in cells depleted of RNF126 or Rabring7 have a comparable luminal pH and distribution compared to control cells but may be compromised in the formation of intraluminal vesicles. EGF-induced multivesicular bodies are decreased in RNF126- and Rabring7-knockdown lines

The destabilization of ESCRT-II as well as the decrease in SCAMP3 phosphorylation suggested that cells depleted of RNF126 or Rabring7 may have defects in the formation of multivesicular bodies. To investigate this directly, we employed

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Fig. 7. Depletion of RNF126 alters the EGFR sorting machinery. (A) Knockdown of RNF126 prolongs the association between c-Cbl and the EGFR. Non-silencing and RNF126-knockdown lines were stimulated for the indicated time and c-Cbl was immunoprecipitated. The immunoprecipitate was analyzed by serially immunoblotting with antibodies against EGFR and c-Cbl. (B) EGF-induced phosphorylation of the RNF126-interacting protein YMER is decreased in cells depleted of RNF126. RNF126 associates with YMER (left panel). HEK-293T cells transfected with myc-YMER were lysed and incubated with either GST or GST–RNF126, and bound material was analyzed by immunoblotting with an antibody against myc. YMER phosphorylation is decreased in the RNF126-knockdown line (right panel). YMER was immunoprecipitated from both control and RNF126-knockdown lines and the immunoprecipitate was analyzed by serially immunoblotting with an anti-phosphotyrosine antibody and anti-YMER. (C) Co-depletion of Vps22 in the RNF126- and Rabring7-knockdown lines. Lysate from the non-silencing, RNF126knockdown lines and the Rabring7-knockdown lines were analyzed with antibodies against HRS, TSG101, Vps22 and Vps24. (D) EGF-induced SCAMP3 phosphorylation is decreased in RNF126-knockdown lines. GST–RNF126 associates with SCAMP3 in a ligand-independent manner (left panel). Immobilized GST or GST– Rabring7 were incubated with cell lysate derived from HeLa cells that were serum starved and either left unstimulated or stimulated with EGF for 2 min. Bound material was analyzed with an antibody against SCAMP3. Knockdown of either RNF126 reduces SCAMP3 phosphorylation (right panel). SCAMP3 was immunoprecipitated from control and RNF126-knockdown lines. The immunoprecipitate was analyzed by serially immunoblotting with antibodies against phosphotyrosine and SCAMP3.

cryo-electron microscopy and quantified the number of multivesicular bodies observed in either control or knockdown lines after 30 min EGF stimulation. For each cell line, we examined 20 fields of view between two replicate experiments and counted the number of multivesicular bodies defined as being unilaminar vacuoles greater the 200 nm in width that contained one or more internal vesicles. In the control cells we counted 24 of these structures while only five were observed in the RNF126knockdown and six in the Rabring7-knockdown lines (Fig. 8) suggesting that cells depleted of RNF126 and Rabring7 have fewer multivesicular bodies after EGF stimulation. Discussion Here we provide evidence for the involvement of RNF126 and Rabring7 in the downregulation of the EGFR. We show that

RNF126 and Rabring7 associate with the EGFR, that the receptor is a substrate for RNF126 and Rabring7 mediated ubiquitylation, and that depletion of RNF126 and Rabring7 alter the pattern of EGFR ubiquitylation. Numerous publications have established that c-Cbl mediated EGFR ubiquitylation is involved in receptor downregulation (Levkowitz et al., 1999; Levkowitz et al., 1998). However, the effect of c-Cbl knockdown, or mutations that uncouple EGFR from c-Cbl differs from the effects we observed in RNF126- or Rabring7-knockdown cells with respect to EGFR degradation. In cells depleted of c-Cbl, the EGFR is retained in an early endocytic compartment where it is capable of recycling back to the plasma membrane (Duan et al., 2003; Grøvdal et al., 2004; Ravid et al., 2004). In cells depleted of either RNF126 or Rabring7, however, the EGFR progresses normally through the early endosome but is retained in a late endocytic compartment.



Fig. 8. Cells depleted of RNF126 or Rabring7 have fewer multivesicular bodies. Control, RNF126- or Rabring7-knockdown lines were stimulated with EGF for 30 min and processed for cryo-electron microscopy. Twenty fields of view were examined from two replicate experiments and multivesicular bodies (indicated by arrowheads) were counted in each field of view. A total of 24 multivesciular bodies were identified in the non-silencing line, 5 in the RNF126-knockdown line and 6 in the Rabring7-knockdown line.

We suggest a model where c-Cbl mediated EGFR ubiquitylation leads to the subsequent recruitment of RNF126 and Rabring7 through their ubiquitin-binding zinc fingers. Continued ubiquitylation by RNF126 and Rabring7 may be important in sorting the receptor towards the lysosome for degradation. Indeed, persistent EGFR ubiquitylation has been shown to correlate with lysosomal degradation of the receptor (Huang et al., 2006; Longva et al., 2002; Roepstorff et al., 2009). There have been conflicting reports as to whether the overexpression of Rabring7 accelerates or inhibits EGFR degradation (Mizuno et al., 2003; Sakane et al., 2007). We show that like RNF126, Rabring7 associates with and ubiquitylates the EGFR and that the depletion of Rabring7 delays EGFR degradation suggesting that Rabring7 is also a negative regulator of EGFR. In support of our model we show that RNF126 and Rabring7 associate with the EGFR

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through a ubiquitin-binding domain and that the association between the EGFR and RNF126 or Rabring7 is reduced in cells lacking c-Cbl or in cells expressing a dominant negative form of c-Cbl. RNF126 mediates the formation of K63-linked chains with the E2 Ubc13/Uev1a. Previous work has shown that the EGFR is primarily modified with K63-linked ubiquitin while the E3-E2 complex that drives the formation of these chains remains to be identified (Huang et al., 2006). Therefore, one potential function of RNF126 might be to produce a qualitative change in EGFR ubiquitin chain topology. We observed that the knockdown of either RNF126 and Rabring7 affects the stability of ESCRT-II and that cells depleted of these E3s also contain fewer multivesicular bodies after EGF stimulation. While we cannot rule out the possibility that global changes to the ESCRT machinery and multivesicular body formation contribute to the EGFR sorting defect, our observations are consistent with RNF126 and Rabring7 functioning in cargo specific sorting events. RNF126 depletion had more pronounced effects on EGFR degradation than Rabring7, and co-depletion did not further delay the degradation of the EGFR. Furthermore, RNF126 and Rabring7 depletion had different effects on two additional receptors that are ubiquitylated and sorted by ESCRT. While RNF126 and Rabring7 depletion disrupted the sorting of CXCR4, only depletion of Rabring7 disrupted degradation of MET. We are currently investigating whether CXCR4 or MET are direct substrates of RNF126 or Rabring7. These observations support a model in which RNF126 and Rabring7 regulate the sorting of specific ubiquitylated cargo rather than playing a role in the endosome maturation program. In agreement, the knockdown of RNF126 or Rabring7 did not affect the distribution and intensity of the late endosomal markers CD63 and LBPA or the volume of acidic LysoTracker-positive compartments. We also fail to observe any physical association between GST–RNF126 and GST–Rabring7 with the ESCRT machinery. Previous work has shown that EGF stimulation increases inward budding and the number of multivesicular bodies (White et al., 2006). Furthermore, an EGFR mutant that can not be ubiquitylated fails to engage the ESCRT machinery and induce inward budding at the late endosome (Eden et al., 2012). In Saccharomyces cerevisiae cargo ubiquitylation is required for multivesicular body formation (Macdonald et al., 2012) and strains defective in K63-linked ubiquitylation show changes in the structure of multivesicular bodies (Erpapazoglou et al., 2012). Therefore, an interesting possible explanation for our observations is that RNF126 and Rabring7, through their ubiquitin-binding zinc finger domains, might associate with multiple cargo modified by another E3 and regulate their progression through the endocytic system. RNF126 and Rabring7 also differ in with respect to their ligase activity in vitro. RNF126 uniquely supports ubiquitylation with Ubc13/Uev1a. Both RNF126 and Rabring7 are productive with UbcH5b; however, RNF126 but not Rabring7 mediate the specific formation of K48-linked chains with this E2. We show that the selective formation of K48-linked chains in the reactions containing RNF126 and UbcH5b is dependent on the ubiquitinbinding zinc finger of RNF126. One possible explanation is that the zinc finger orients the acceptor ubiquitin on the growing chain such that only the lysine at position 48 is conjugated to the successive ubiquitin. We are currently investigating how the structural differences between RNF126 and Rabring7 relate to the

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differences in ligase function and whether these differences are recapitulated in cells. In conclusion, our results indicate that both RNF126 and Rabring7 are involved in the sorting of the EGFR at the late endosome. We suggest that RNF126 and Rabring7 are involved in the trafficking of multiple cargo that pass through the late endosome and we are currently investigating other potential substrates for RNF126 and Rabring7. These findings further our understanding regarding the role of ubiquitin in degradative protein traffic. Materials and Methods


cDNA constructs and mutagenesis

Full length murine RNF126 cDNA was cloned into pGEX4T3 (BamHI/EcoRI), pcDNA 3.1 and HA-pcDNA 3.1. The C231A and C13,16A point mutations were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene). The RNF126 truncation mutants used in this study were: the isolated zinc finger (amino acids 1–63), the isolated RING finger (amino acids 208–292) and the DZnF (amino acids 48–313). Full-length murine Rabring7 was also cloned into pGEX4T3 (EcoRI/ XhoI) and the C229A and C22,25A mutations were generated using QuikChange. The Rabring7 truncation mutants used in this study were: the isolated zinc finger domain (amino acids 1–69) and the isolated RING finger domain (amino acids 206– 290). The cytoplasmic regions of FLT3 and EGFR were amplified by PCR and cloned into pcDNA 3.1. EGFR-FLAG in pcDNA 3.1 was a gift from Michael Moran, The Hospital for Sick Children, Toronto, Canada; HA-UB was a gift from Yossef Yarden, Weizmann Institute of Science, Rehovot, Israel; and HA-CXCR4 was a gift from Adriano Marchese, Loyola University, Chicago, USA. YMER was obtained from the human ORFeome and cloned into pDEST-nMYC using the Gateway system (Invitrogen, Burlington, CA, USA). Cell culture and generation of stable knockdown lines

HeLa cells were maintained in DMEM supplemented with 10% FBS. Clonal lines were generated by transfecting cells with 3 mg of pGIPZ shRNA (Open Biosystems, Lafayette, CO, USA) with a non-silencing sequence or with sense strand sequence against RNF126: 59-CACTCAAACCCTATGGACT-39 or against Rabring7: 59-TGTATGTAGGAAGAGCTTA-39 or 59-GGGTTTAGAGTGTCCAGTA-39. Following selection in 1 mg/ml puromycin, colonies were isolated and propagated to obtain a clonal line. The generation of HEK-293 cells that express EGFR–FLAG (HEK-EGFR) was previously described (Tong et al., 2008). Polyclonal cell lines were generated by transfecting cells with the pGIPZ constructs described above. Cells were selected in 1 mg/ml puromycin and 400 mg/ml G418. For transient knockdown of RNF126, HeLa cells seeded in six-well dishes were transfected with 20 pmol siRNA targeting RNF126: 59-CCGGATTATATCTGTCCAAGA-39 (Qiagen, Valencia, CA, USA) or an Allstars negative control siRNA (Qiagen) using Lipofectamine 2000. Experiments were conducted 48 hours post-transfection. For stimulation of HeLa cells with EGF ligand, cells were serum starved overnight in DMEM, washed in PBS, and DMEM containing 20 ng/ml human recombinant EGF (R&D Systems, Minneapolis MN, USA) was added for the indicated time points. Ligand stimulation of HEK-EGFR cells was performed similarly with the exception that 100 ng/ml of EGF was used. For stimulation with HGF, cells were serum starved overnight in DMEM, washed in PBS, and DMEM containing 100 mg/ml cycloheximide (Sigma) was added for 2 hours to prevent de novo protein synthesis during the course of the experiment. Medium containing 20 ng/ml human recombinant HGF (Sigma) and 100 mg/ml cycloheximide was added for the indicated time before lysis in RIPA buffer. For stimulation with SDF1a, control and knockdown lines were transfected with 3 mg of HA-CXCR4 and split onto glass coverslips. 24 hours post-transfection, cells were serum starved for 5 hours in DMEM containing 100 mg/ml cycloheximide. Medium containing 240 ng/ml human recombinant SDF-1a (Peprotech, Rocky Hill, NJ, USA) and 100 mg/ml cycloheximide was added for 3 hours before fixation. WT and c-Cbl2/2 murine embryonic fibroblasts (a gift from Hamid Band, University of Nebraska, Omaha, USA) were maintained in DMEM supplemented with 10% FBS. For stimulation, cells were serum starved overnight in 0.5% FBS DMEM, washed in PBS, and DMEM containing 100 ng/ml murine EGF (Millipore) was added for the indicated time period before lysis. Ba/F3 expressing wild-type FLT3 (Ba/F3-FLT3 cell line) were kindly provided by Robert Rottapel. Ba/F3-FLT3 cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and 5% WEHI cell supernatant (as a source of IL3). For stimulation with FLT3 ligand, cells were starved of IL-3 for 5 hours, suspended in serum-free RPMI at 256106 cells/ml and 100 ng/ml mFLT3 ligand (R&D Systems) at 37 ˚C for various times.

sonicated in PLC lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton-x-100, 1 mM EGTA (pH 8.0) freshly supplemented with complete protease inhibitors (Roche Applied Science, Laval, QC, Canada). The cleared bacterial lysate was incubated for 30 min at 4 ˚C with glutathione– Sepharose (GS) beads (GE Healthcare, Cooksville, ON, Canada) which were then washed four times with PLC. For elution of the fusion proteins the GS bound material was washed an additional three times with PBS followed by three washes with 50 mM Tris-HCl (pH 7.5). The fusion proteins were eluted in Tris-HCl with 10 mM reduced glutathione (pH 8.0). In vitro ubiquitylation assay

Ubiquitylation assays were performed in a 25 ml reaction volume with the following components: 0.18 mM E1 from yeast (Boston Biochem, Cambridge, MA, USA), either 0.1 mM UbcH5b or 0.1 mM UbcH13/Uev1a (Boston Biochem), 1.16 mM of GST fusion protein (E3), 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 0.2 mM dithiothreitol, 1 mM ATP, and ubiquitin (11.6 mM). The reaction was incubated at 30 ˚C for 90 min and stopped by adding 26 SDS sample buffer. The mixture was resolved by SDS-PAGE and analyzed by western blot using mouse anti-ubiquitin monoclonal antibody (Covance). [35S]Methionine-labeled FLT3 and EGFR were in vitro translated using the TNT Coupled Wheat Germ extract system (Promega, Madison, WI, USA). Aliquots were added to the in vitro ubiquitylation assays described above and analyzed by SDS-PAGE and autoradiography. Immunoprecipitations, in vitro binding assays and western blotting

For western blotting, lysates were prepared in RIPA buffer (150 mM NaCl, 20 mM Tris (pH 7.5), 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and boiled in SDS-Laemmli sample buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Pall Corp, Mississauga, ON, Canada). Quantification was performed by chemiluminescence using the Alpha Innotech Fluorochem 8000 imaging system (Santa Clara, CA, USA). A two-tailed unpaired t-test was used to generate P-values using GraphPad Prism. For immunoprecipitation and GST-mixing experiments, cells were lysed in Nonidet P-40 lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Nonidet p-40, 1 mM EGTA (pH 8.0)] supplemented with complete protease inhibitors (Roche Applied Science), 1 mM sodium orthovanadate and 10 mM NaF. Lysates were cleared by centrifugation (14,000 rpm for 20 min) pre-cleared by incubation with protein-A– or protein-G–Sepharose beads for 30 min at 4 ˚C with gentle agitation. Precleared lysates were incubated with antibodies and either protein-A– or protein-G–agarose beads at 4 ˚C for 2 hours with gentle agitation, washed three times in 1 ml of cold NP-40 wash buffer and proteins were eluted by boiling in SDS-Laemmli sample buffer. For GST-mixing experiments lysates were incubated with 5 mg of fusion protein for 2 hours and washed with NP40 wash buffer before SDS-PAGE. To examine EGFR ubiquitylation, transfected HEK-293T cells were lysed in boiling RIPA buffer containing 1% SDS and 10 mM N-ethylmaleimide (Sigma, Oakville, ON, CA), passed through a syringe and diluted 10-fold before immunoprecipitation as described above. Antibodies and reagents

RNF126 and Rabring7 antibodies were produced by immunizing rabbits with peptides corresponding to the C-terminal regions of each protein, Rabring7: CNRFSNDSQLHDRWTF, RNF126: CSPSNENATSNS. 5 ml of crude serum was used for immunoprecipitation and a 1:500 dilution was used for western blotting. RNF126 and Rabring7 antisera immunoprecipitated a single band in each case (supplementary material Fig. S1). The K48 and K63 tetra-ubiquitin chains were from Boston Biochem (Boston, MA, USA). LysoTracker Red DND-99 was from Invitrogen (Burlington, CA, USA). The following commercial antibodies were used: TSG101 (Santa Cruz Biotechnology, mouse, Santa Cruz, CA, USA); Vps22 (AbD Serotec, mouse, Raleigh, NC, USA); YMER (Sigma, rabbit); Vps24 (Sigma, rabbit); FLAG-M2 (Sigma, mouse); b-tubulin (Sigma, mouse); EGFR (for immunoprecipitation and western blotting, Millipore, rabbit, Billerica MA, USA); phosphotyrosine (Millipore, mouse); EGFR (for immunocytochemistry, Cell Signaling, rabbit, Danvers MA, USA), HA (Roche, mouse); HA (for immunofluorescence, Novus Biologicals, rabbit, Littleton, CO, USA); ubiquitin (Covance, mouse, Princeton, NJ, USA); CD63 (Developmental Studies Hybridoma Bank, University of Iowa); EEA1 (BD Transduction Labs, mouse, Franklin Lakes, NJ, USA); c-Cbl (BD Transduction Labs, mouse); HRS (a gift from Sylvie Urbe, University of Liverpool, Liverpool, UK) and SCAMP3 (R&D Systems, goat); Rab7 (Cell Signaling, rabbit); GFP (Clontech, rabbit, Mountain View, CA, USA); MET (Santa Cruz Biotechnology, mouse, Santa Cruz, CA, USA); LBPA (Echelon Biosciences, mouse, Salt Lake City, UT, USA). The following secondary antibodies were used: HRP-linked anti-mouse IgG (GE Healthcare, sheep); HRP-linked protein A (GE Healthcare) and HRP-linked anti-rabbit (Cell Signaling, goat).

Production of GST fusion proteins

Immunocytochemistry

pGEX transformed BL21 were induced with 0.3 mM isopropyl-1-b-D-thiogalactopyranoside (IPTG) at 18 ˚C for 16 hours. The bacterial pellets were

HeLa cells were seeded on glass coverslips and fixed in a 2% PFA, 30 mM sucrose for 30 min at room temperature, incubated in 100 mM glycine to quench the PFA,


RNF126 and Rabring7 regulate endosomal sorting of EGFR permeabilized with 0.1% TX-100 and blocked in a 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA). Coverslips were inverted on primary antibody for 30 min in a humidified chamber at 37˚C. Cells were washed 36 for 10 min in 0.05% TX-100 and primary antibodies indirectly labeled for 30 min at 37˚C with: Cy5-conjugated donkey anti-mouse IgG and Cy3-conjugated donkey anti-rabbit IgG from Jackson ImmunoResearch laboratories. Cells were washed again for 36 10 min in 0.05% Triton X-100 and mounted in Dako Cytomation fluorescent mounting medium. Images were acquired using a Zeiss Axiovert 200 inverted fluorescence microscope using either 606/1.35NA or 636/1.4NA objectives equipped with a back-thinned EM charged-coupled device camera (Hamamatsu, Bridgewater, NJ, USA), and a spinning disk confocal scan head. Equipment was driven by Volocity (PerkinElmer, Waltham, MA, USA) acquisition software. Linear adjustments for contrast and brightness were performed using the Volocity software. To measure the volume of vesicular Cy3 (EGFR), we first determined the minimum intensity of the Cy3 punctae. The volume of positivity (mm3) above this threshold (5000 AU) was determined for each image and qualified as vesicular EGFR. Next, the total cellular volume was determined. As the pGIPZ constructs contain an IRES eGFP, all stable cell lines used are GFP positive. The software automatically determined the volume of GFP (mm3) which was considered total cellular volume. The ratio of Cy3 above threshold to total GFP is reported. Four randomly chosen fields of view for each condition were examined. The volume measurements for LysoTracker-positive compartments were performed similarly where five fields of view were examined under each condition. For colocalization analysis of EGFR with EEA1 and CD63, the Cy5 channel was first reassigned to green and images were deconvolved using the fast restorative function in Volocity. A point spread function was applied to each channel with the following parameters: Numerical aperture51.2, medium refractive index51.33 and emission wavelengths of 561 nm (Cy3) and 639 nm (Cy5). The minimal intensity threshold to be included in the colocalization measurement was determined by measuring five regions of interest within the cell outside of any specific punctate structures. Pearson’s correlation was measured on $5 individual cells under each condition cells using the colocalization function in Volocity. To measure the volume of vesicular HA–CXCR4 (Cy3), we determined the minimum intensity of the Cy3 punctae (15,000 AU) and measured the volume (mm3) above this threshold relative to the total Cy3 volume in each image. Eight randomly chosen fields of view for each condition were examined. For colocalization analysis, the Cy5 channel was reassigned to green. The minimal intensity threshold to be included in the colocalization measurement was determined by measuring 5 ROIs within the cell outside of any specific punctate structures. Images were deconvolved using the fast restorative function in Volocity. A point spread function was applied to each channel with the following parameters: Numerical aperture51.3, medium refractive index51.33 and emission wavelengths of 561 nm (Cy3) and 639 nm (Cy5). The minimal intensity threshold to be included in the colocalization measurement was determined by measuring 5 ROIs within the cell outside of any specific punctate structures. Mander’s coefficient was measured on eight randomly chosen cells for each condition using the colocalization function in Volocity. Two tailed unpaired t-tests were performed to generate P-values using GraphPad Prism. Electron microscopy

Cell pellets were high-pressure frozen with a Leica HPM100 (Concord ON, Canada), freeze-substituted with 1% osmium tetroxide and 0.1% uranyl acetate in acetone with a Leica AFS2, and embedded in a Quetol-Spurr resin mixture. Sections 100 nm thick were cut with a RMC MT6000, stained with uranyl acetate and lead citrate and viewed with a FEI Tecnai20 TEM (Hillsboro, OR, USA).

Acknowledgements

We thank all of the members of the McGlade lab for helpful discussion and insight. We thank Micheal Woodside and Paul Paroutis for assistance with the imaging and Doug Holmyard for performing the electron microscopy experiments and imaging. We thank the following people for the gift of reagents: Hamid Band for the c-Cbl2/2 murine embryonic fibroblasts, Michael Moran for the EGFR-FLAG construct and HEKEGFR cells, Yossi Yarden for the HA-UB construct, Adriano Marchese for the HA-CXCR4 construct and Sylvie Urbe for the HRS antibody. Author contributions

C.J.M. conceived the study. C.J.S. and C.J.M. designed the experiments, analyzed the data and prepared the manuscript. C.J.S. performed the experiments. D.M.B. performed the initial studies on RNF126. Funding

This work was supported by a Canadian Institutes for Health Research [grant number MOP 12859 to C.J.M.]; a studentship from

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the Hospital for Sick Children Research Training Center to C.J.S.; and a Vanier Canada Graduate Scholarship to C.J.S. Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.116129/-/DC1

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