ABSTRACT
We and others have shown that trafficking of G-protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Here, we show by overexpression and depletion studies that HACE1, a HECT-domain-containing ubiquitin ligase, promotes the recycling of the β2-adrenergic receptor (β2AR), a prototypical G-protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the β2AR in conjunction with HACE1 triggered ubiquitylation of Rab11a, as observed by western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitylated on Lys145. A Rab11a-K145R mutant failed to undergo β2AR–HACE1-induced ubiquitylation and inhibited the HACE1-mediated recycling of the β2AR. Rab11a, but not Rab11a-K145R, was activated by β2AR–HACE1, indicating that ubiquitylation of Lys145 is involved in activation of Rab11a. Co-expression of β2AR–HACE1 also potentiated ubiquitylation of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases through their ubiquitylation, with associated functional effects demonstrated on Rab11a. This suggests a new pathway whereby a cargo protein, such as a G-protein-coupled receptor, can regulate its own trafficking by inducing the ubiquitylation and activation of a Rab GTPase.
INTRODUCTION
G-protein-coupled receptors (GPCRs) represent ∼4% of the human genome and form one of the largest and most studied family of proteins (Fredriksson and Schiöth, 2005; Harrow et al., 2012; Venter et al., 2001). They mediate physiological responses to a vast array of cellular mediators such as hormones, neurotransmitters, lipids, nucleotides, peptides, ions and photons. All GPCRs share a common molecular topology with a hydrophobic core of seven transmembrane α-helices, three intracellular loops, three extracellular loops, an extracellular N-terminus and an intracellular C-terminus. GPCRs are typically delivered to the plasma membrane in a ligand-responsive and signaling-competent form. Following agonist stimulation, the majority of GPCRs internalize into endosomes and can then undergo recycling to the cell surface or lysosomal degradation (Costanzi et al., 2009; Pierce et al., 2002; Ritter and Hall, 2009). The fact that more than 30% of prescribed drugs target GPCRs highlights their importance in the treatment of disease (Hopkins and Groom, 2002). Therefore, a better comprehension of the cellular events controlling their intracellular trafficking is essential to improve the actual drug efficacy and specificity, but also to identify new pharmaceutical targets.
Our laboratory and others have previously characterized Rab GTPases as key regulators of GPCR trafficking (Hamelin et al., 2005; Lachance et al., 2011; Parent et al., 2009; Seachrist et al., 2002; Wikström et al., 2008; Zhang et al., 2009). More than 60 Rab GTPases, forming the largest branch of Ras-related small GTPases, are involved in almost every step of vesicle-mediated transport (Zerial and McBride, 2001). Each Rab GTPase has a distinct subcellular localization that correlates with the compartments between which they coordinate transport (Hutagalung and Novick, 2011). These proteins are well-recognized targets in human disease, but to date are underexplored therapeutically. Elucidation of how dysregulated Rab proteins and accessory proteins contribute to disease remains an area of intensive study and an essential foundation for effective drug targeting. Cancer, neurodegeneration, diabetes and bone diseases represent examples of pathologies resulting from aberrant function of Rab GTPases (Kelly et al., 2012; Li, 2011; Richards and Rutherford, 1990).
Like other GTPases, Rab proteins shuttle between the inactive (GDP-bound) and active (GTP-bound) conformations. To do so, distinct regulators promote the exchange of GDP to GTP (guanine nucleotide exchange factors, GEFs) and GTP hydrolysis (guanine nucleotide activating proteins, GAPs) (Schwartz et al., 2007). Despite the large number of Rabs, very few GEFs and GAPs have been identified for these small GTPases to date. To cite a few, some DENN (differentially expressed in normal and neoplastic cells) domain proteins such as Rab6IP1 (a Rab39 GEF), and Vsp9 domain proteins such as Rabex5 (a Rab5 GEF), have been described as Rab GEFs. However, Rab GAPs are known to share a common TBC1 (Tre-2/Bub2/Cdc16) domain (Barr and Lambright, 2010; Marat et al., 2011; Xiong et al., 2012). It has been shown that the GPCR angiotensin II type 1A receptor (AT1AR) increases the GTP-binding of Rab5a. This study not only reported the first evidence that GPCRs might control activity of Rabs, but it also underlined that a direct interaction between the receptor and the GTPase seems necessary for this effect (Seachrist et al., 2002). However, it remains unclear whether the receptor itself acts as a GEF or recruits proteins possessing a GEF activity.
It was recently described that mono-ubiquitylation enhances activation of K-Ras and facilitates its binding to specific effectors (Sasaki et al., 2011). Considering the small number of characterized Rab GEFs, and the size of the Rab family, one could speculate that activation of Rab GTPases might also be controlled by a general mechanism involving post-translational modifications such as ubiquitylation. However, Rab ubiquitylation has not been described so far. Interestingly, HACE1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1) was identified as a Rab interactor (Tang et al., 2011) but no effect on Rab ubiquitylation was reported. Because β2-adrenergic receptor (β2AR) trafficking is regulated by various Rab proteins (Awwad et al., 2007; Dong and Wu, 2007; Dong et al., 2010; Hammad et al., 2012; Moore et al., 2004; Parent et al., 2009; Seachrist et al., 2000), we investigated whether HACE1 was able to modulate β2AR trafficking. Here, we report that co-expression of β2AR with HACE1 induces the ubiquitylation and activation of Rab11a, which in turn regulates β2AR recycling. Ubiquitylation of other specific Rabs was also observed in the presence of β2AR–HACE1. Together, our data uncover a new regulatory mechanism for Rab GTPases by which a cargo protein can direct its own trafficking.
Results
HACE1 interacts with and regulates the recycling of the β2AR
Tang and colleagues reported that Rab1, Rab4 and Rab11 associate with HACE1 (Tang et al., 2011). Because these GTPases are known to regulate specific events in β2AR trafficking (Hammad et al., 2012; Parent et al., 2009; Seachrist et al., 2000), we assessed whether the receptor could also interact with HACE1. Immunoprecipitation of HA-tagged β2AR stably expressed in HEK293 cells revealed that endogenous HACE1 co-immunoprecipitated with the receptor (Fig. 1A). The effect of HACE1 on trafficking of transiently expressed β2AR was then determined by ELISA. Transient expression of Myc-HACE1 and Myc-HACE1-C876S (a mutant with deficient E3-ligase activity) (Anglesio et al., 2004; Tang et al., 2011; Zhang et al., 2007) increased cell surface expression of β2AR by roughly 25%, indicating that the catalytic activity of the enzyme is not required for this effect (Fig. 1B). Time-course analyses established that HACE1 significantly decreased the apparent agonist-induced internalization of the β2AR, whereas HACE1-C876S had virtually no effect (Fig. 1C). Because an increase in the receptor recycling could explain these results, internalization assays were carried out in the presence of a recycling inhibitor, monensin (Hamelin et al., 2005). As can be seen in Fig. 1D, treatment with monensin inhibited the effect of HACE1 on internalization of β2AR, indicating that HACE1 regulates β2AR recycling. This was further studied by recycling time-course experiments in cells treated with 5 µM isoproterenol for 15 minutes at 37°C, and then incubated in DMEM containing 10 µM propranolol for the indicated time periods to prevent further receptor internalization and to allow receptor recycling (Fig. 1E). Data obtained confirmed that HACE1 promoted β2AR recycling, whereas HACE1-C876S did the opposite after 60 minutes of recycling (Fig. 1E). We were then interested in verifying the endogenous colocalization of HACE1 with β2AR. To do so, colocalization studies were carried out using cells stably expressing HA-β2AR. Comparative analyses revealed no significant differences between the trafficking properties of transiently expressed β2AR compared with stably expressed β2AR (supplementary material Fig. S1). Both systems were thus used interchangeably in the present report. As shown in Fig. 1Fa–d, HACE1 and β2AR colocalize into intracellular compartments found throughout the cytoplasm, in the proximity of the cell membrane and in the perinuclear region, under basal conditions (Fig. 1Fd, extracted colocalizing pixels). Agonist stimulation of the receptor resulted in internalization of the β2AR from the plasma membrane into intracellular compartments and in a distribution of HACE1 that concentrated towards the perinuclear region where it predominantly colocalized with the receptor (Fig. 1Ff–i). The intracellular distribution of HACE1 and its colocalization with the receptor was similar to that seen in basal conditions as receptor recycling progressed (Fig. 1Fk–s).
HACE1 interacts with β2AR and regulates its cell surface expression and recycling. (A) Immunoprecipitation (IP) was performed using a HA-specific monoclonal or isotypic IgG1 control antibody on lysates from HEK293 cells stably expressing HA-β2AR and immunoblotting (IB) was carried out with an HACE1-specific polyclonal antibody. The blots shown are representative of three separate experiments. (B) HEK293 cells were cotransfected with FLAG-β2AR and pcDNA3, wild-type Myc-HACE1 or Myc-HACE1-C876S. Expression of cell surface receptor was measured by ELISA using a FLAG-specific monoclonal antibody. (C) Cells were treated with 5 µM isoproterenol for the indicated time periods. Quantification of surface receptors was performed by ELISA and the percentage of receptor internalization was calculated. (D) Cells were pre-treated with 25 µM monensin (a recycling inhibitor) or ethanol (vehicle) for 30 minutes at 37°C, and then stimulated with 5 µM isoproterenol for 60 minutes at 37°C in the presence of monensin. Cell surface receptors were measured by ELISA and the percentage of receptor internalization was calculated. (E) Cells were treated with 5 µM isoproterenol for 15 minutes at 37°C, and then incubated in DMEM containing 10 µM propranolol for the indicated time periods to prevent further internalization and to allow receptor recycling. Receptor cell surface expression was detected by ELISA and the percentage of receptor recycling was calculated. Results are means ± s.e.m. of at least five separate experiments. The statistical significance was determined using paired and unpaired Student's t-tests (B,C) or regular two-way ANOVA test (D–E) followed by Bonferroni post-tests. *P<0.05, **P<0.01,***P<0.001. (F) Colocalization analyses of endogenous HACE1 in cells stably expressing HA-β2AR. Cells were treated with 5 µM isoproterenol for 15 minutes at 37°C, and then incubated in DMEM containing 10 µM propranolol for the indicated time periods to prevent further internalization and to allow receptor recycling. Cells were then fixed, permeabilized and labeled with rabbit polyclonal anti-HACE1 antibody. The third image on the right represents a merged image of the red-labeled β2AR (a,f,k,p) and green-labeled HACE1 (b,g,l,q) signals where the areas with a high degree of colocalization appear in yellow (c,h,m,r). Scale bars: 10 µm. Colocalizing pixels (d,i,n,s) and fluorograms illustrating HA-β2AR colocalization with HACE1 (e,j,o,t) are presented.
The functional implication of endogenous HACE1 on the trafficking of stably expressed β2AR was assessed in cells transfected with HACE1 siRNA. Depletion of HACE1 significantly reduced the cell surface expression of the β2AR compared with expression in control cells (Fig. 2A). There was a ∼33% increase in apparent agonist-induced internalization of the receptor when cells were transfected with HACE1 siRNA compared with cells transfected with control siRNA (60% compared with 45% internalization, respectively) (Fig. 2B). β2AR recycling after agonist-induced internalization was inhibited by ∼25% in cells depleted of HACE1 compared with the control (Fig. 2C). These data validated the results obtained with HACE1 overexpression and established a new role for HACE1 in β2AR cell surface expression and recycling.
Depletion of HACE1 affects β2AR trafficking. HEK293 cells stably expressing HA-β2AR were transfected with negative control (CTRL) or siRNA against HACE1 for 72 hours and cell surface receptor expression (A), the percentage of receptor internalization after stimulation with 5 µM isoproterenol for 15 minutes (B), and receptor recycling after stimulation with 5 µM isoproterenol for 15 minutes at 37°C, and incubation in DMEM containing 10 µM propranolol for 60 minutes (C) was measured by ELISA using a HA-specific monoclonal antibody. Efficiency of HACE1 depletion by siRNA was confirmed by western blot analysis with a HACE1-specific antibody (A, inset). Results are mean ± s.e.m. of three to five separate experiments. The statistical significance was determined using a regular two-way ANOVA test followed by Bonferroni post-tests. *P<0.05, **P<0.01.
HACE1 regulates β2AR recycling through Rab11a
Previous studies showed that β2AR recycling is regulated by Rab4 and Rab11 (Moore et al., 2004; Parent et al., 2009; Seachrist et al., 2000). Because HACE1 is reported to associate with Rab4 and Rab11 (Tang et al., 2011), we were thus interested in determining whether HACE1 regulated β2AR recycling through one of these two GTPases. β2AR recycling was thus measured in cells cotransfected with different combinations of HACE1, Rab4a or Rab11a (Fig. 3A). Transfection of HACE1 strongly promoted receptor recycling compared with levels in cells transfected with pcDNA3. Co-expression of Rab4a or Rab11a did not have any significant effect on β2AR recycling. Interestingly, β2AR recycling in the presence of HACE1 was strongly enhanced by Rab11a co-expression whereas cotransfection of Rab4 prevented the HACE1-mediated effect on β2AR recycling. This could be possibly explained by the ability of Rab4a to interact with HACE1 (Tang et al., 2011) and to compete with other effectors involved in β2AR recycling through HACE1 in our system. Fig. 3B shows that depletion of endogenous Rab11a with siRNA significantly reduced the recycling of the β2AR when expressed alone and inhibited the HACE1-mediated increase in β2AR recycling by 40%. We show in Fig. 1F that β2AR colocalizes intracellularly with endogenous HACE1 and it has been shown previously to colocalize with Rab11 (Parent et al., 2009). Confocal microscopy studies showed that endogenous HACE1 and HA-Rab11a, expressed at low levels to reflect distribution of endogenous Rab11a, colocalized mostly in perinuclear intracellular compartments, but also in peripheral intracellular vesicles in HEK293 cells (Fig. 3C). Fluorogram analysis revealed that there is a high degree of colocalization between HACE1 and Rab11a. Colocalization between β2AR-GFP, endogenous HACE1 and Rab11a was also detected in perinuclear compartments (Fig. 3Cd, inset).
HACE1 regulates β2AR recycling through Rab11a. (A) HEK293 cells were transiently cotransfected with FLAG-β2AR and pcDNA3, Myc-HACE1, HA-Rab4a, HA-Rab4a + Myc-HACE1, HA-Rab11a, or HA-Rab11a + Myc-HACE1. Cells were treated with 5 µM isoproterenol for 15 minutes at 37°C, and then incubated in DMEM containing 10 µM propranolol for 15 minutes to prevent further internalization and to allow receptor recycling. Expression of cell-surface receptor was detected by ELISA and the percentage of receptor recycling was calculated. Results are means ± s.e.m. of five separate experiments. (B) HEK293 cells stably expressing HA-β2AR were pretreated with negative control (siCTRL) or siRNA against Rab11a for 24 hours and then cotransfected with pcDNA3 or Myc-HACE1. Cells were treated with 5 µM isoproterenol for 15 minutes at 37°C, and then incubated in DMEM containing 10 µM of propranolol for 60 minutes to prevent further internalization and to allow receptor recycling. Expression of cell-surface receptors was detected by ELISA and the percentage of receptor recycling was calculated. Results are means ± s.e.m. of three separate experiments. Statistical analyses were performed using the regular two-way ANOVA test followed by Bonferroni post-tests (A,B). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (C) HEK293 cells transiently expressing β2AR-GFP were fixed, permeabilized and labeled with rabbit polyclonal anti-HACE1 and mouse anti-HA antibodies. Colocalization of (a) green-labeled β2AR, (b) red-labeled HACE1 and (c) blue-labeled Rab11a appears white in d. Scale bars: 10 µm. Fluorograms illustrating colocalization of β2AR-GFP with HACE1, and colocalization of Rab11a with HACE1 are shown in the bottom two panels.
β2AR promotes HACE1-mediated ubiquitylation of Rab11a
We then evaluated the effects of receptor stimulation as well as of HACE1 or Rab11a co-expression on co-immunoprecipitation of β2AR–HACE1 and β2AR–Rab11a following stimulation with isoproterenol for 0 to 120 minutes of cells expressing the combinations of proteins indicated in Fig. 4A. The interaction between HACE1 and the β2AR was not significantly affected by receptor activation (Fig. 4A, top panel, lanes 4–7). By contrast, β2AR–Rab11a co-immunoprecipitation was down-modulated after 15 and 60 minutes of agonist treatment but returned to basal levels at 120 minutes (Fig. 4A, second panel, lanes 8–11). Of note, HACE1 co-expression caused a significant reduction in the β2AR–Rab11a interaction in the absence of stimulation (t = 0), which was accentuated by receptor activation even up to 120 minutes (Fig. 4A, second panel, lanes 12–15). Densitometry analyses of multiple independent experiments of HACE1 and Rab11a co-immunoprecipitation with the receptor normalized on the receptor immunoprecipitation support these observations (Fig. 4B,C). Our earlier findings showed that the β2AR interacts preferentially with Rab11a in its GDP-bound form (Parent et al., 2009). This, together with the results presented in Fig. 4, could suggest that receptor stimulation and co-expression of HACE1 activates Rab11a, which would result in reduced β2AR–Rab11a interaction.
β2AR modulates Rab11a dimerization and promotes HACE1-mediated ubiquitylation of Rab11a. (A) The β2AR–Rab11a interaction is affected by HACE1 co-expression and receptor stimulation. HEK293 cells were cotransfected with FLAG-β2AR and Myc-HACE1, HA-Rab11a or both proteins and stimulated with 5 µM isoproterenol for the indicated times. Immunoprecipitation (IP) of the receptor was carried out using a FLAG-specific monoclonal antibody. Immunoblotting (IB) was carried out with anti-FLAG, anti-Myc and anti-HA polyclonal antibodies. The blots are representative of three separate experiments. (B,C) Densitometry analyses performed with ImageJ software. The densitometry values of immunoprecipitated Myc-HACE1 or HA-Rab11a were normalized to the amount of immunoprecipitated receptor and reported as a percentage. (D) β2AR expression promotes Rab11a dimerization, which is reversed by the presence of HACE1. HEK293 cells were cotransfected with the indicated constructs. Immunoprecipitation of the GTPases was carried out using an anti-FLAG monoclonal antibody. Immunoblotting was carried out with anti-FLAG, anti-GFP, anti-Myc and anti-HA polyclonal antibodies. (E) Rab11a but not Rab4a is ubiquitylated by the co-expression of β2AR and HACE1. HEK293 cells were cotransfected with the indicated constructs. Immunoprecipitation of the GTPases was performed using anti-HA or anti-FLAG monoclonal antibodies. Immunoblotting was carried out with anti-FLAG, anti-ubiquitin, anti-Myc and anti-HA polyclonal antibodies. The blots shown are representative of three separate experiments. (F) Endogenous Rab11a is ubiquitylated by HACE1. HEK293 cells stably expressing HA-β2AR were treated with negative control (siCTRL) or siRNA against HACE1 for 72 hours. Immunoprecipitation of the GTPase was carried out using anti-Rab11a monoclonal antibodies or isotypic control. Immunoblotting was carried out with anti-Rab11a, anti-ubiquitin, anti-HACE1 and anti-HA polyclonal antibodies. The blots shown are representative of three separate experiments. (G) HACE1 does not ubiquitylate β2AR. HEK293 cells were cotransfected with FLAG-β2AR and the indicated proteins. Immunoprecipitation of the receptor was performed using anti-FLAG monoclonal antibody. Immunoblotting was carried out with anti-FLAG, anti-ubiquitin, anti-Myc and anti-HA polyclonal antibodies. The blots shown are representative of three separate experiments. Molecular masses are indicated on the left.
Interestingly, β2AR and HACE1 expression modified the migration profile of Rab11a (Fig. 4A, see arrows in bottom panel). Indeed, a ∼50 kDa band appeared in lanes 8–11 where the β2AR was co-expressed with Rab11a, suggestive of dimerization of the small GTPase or its association with another protein of ∼25 kDa. This band was not detected in lanes 12–15 where β2AR and HACE1 were co-expressed, which could indicate that the presence of HACE1 decreased Rab11a dimerization or its association with another partner. Instead, a ∼25 kDa Rab11a band and higher bands each heavier by ∼8 kDa were observed in this condition, reminiscent of Rab11a ubiquitylation. To verify whether Rab11 could dimerize in the presence of the receptor, co-immunoprecipitation experiments of differentially tagged HA-Rab11a and FLAG-Rab11a were carried out from lysates of cells cotransfected with empty vector, Myc-HACE1, β2AR-GFP or both proteins (Fig. 4D). Co-expression of β2AR-GFP strongly promoted the FLAG-Rab11a/HA-Rab11a co-immunoprecipitation (Fig. 4D, top panel, lane 6), which was reversed by the co-expression of HACE1 (Fig. 4D, top panel, lane 7). These data suggest that Rab11a can form dimers in cells. Alternatively, our data could also be explained by the association of Rab11a with another protein or with receptor dimers/oligomers binding simultaneously to multiple Rab11a proteins that would be regulated by the receptor and HACE1.
Rab11a ubiquitylation was then assessed in the presence of β2AR, HACE1 or both proteins, in HEK293 cells transfected with the combinations of constructs indicated in Fig. 4E. Ubiquitylation of Rab11a (Fig. 4E, top panel, lane 4), but not of Rab4a (lane 10), was detected in basal conditions in our system. Interestingly, substantial potentiation of Rab11a ubiquitylation was revealed when both the β2AR and HACE1 were co-expressed (lane 8), in contrast to results with Rab4a (lane 14). This was dependent on the E3 ubiquitin ligase activity of HACE1 because HACE1-C876S failed to produce the same effect on Rab11a (lane 9). Furthermore, we observed that depletion of endogenous HACE1 inhibited the ubiquitylation of endogenous Rab11a in cells stably expressing HA-β2AR (Fig. 4F). Since ubiquitylation of GPCRs can regulate their trafficking, we verified whether HACE1 was ubiquitylating the β2AR. As shown in Fig. 4G, HACE1 failed to ubiquitylate the receptor, unlike Nedd4, an HECT-E3-ubiquitin ligase known to be involved in β2AR ubiquitylation (Shenoy et al., 2008). This indicated that HACE1 was not regulating β2AR trafficking by ubiquitylation of the receptor. It is also noteworthy that the shift in mobility reflecting Rab11a ubiquitylation was detected in the presence of HACE1 but not of Nedd4, revealing specificity in this process (Fig. 4G, see arrow on bottom panel).
Ubiquitylation of Lys145 regulates Rab11a GTP loading and recycling of β2AR
To confirm that Rab11a was ubiquitylated and to identify the Lys residue involved, LC-MS/MS was performed on immunoprecipitated HA-Rab11a that was co-expressed with the β2AR in HEK293 cells. Three peptides comprised of the 141AFAEKNGLSFIETSALDSTNVEAAFQTILTEIYR174 amino acids of Rab11a were identified and Lys145 was determined to be ubiquitylated. An HA-Rab11a-K145R mutant was thus generated and its ubiquitylation studied by western blot analysis with a ubiquitin antibody as described in Fig. 5A. Mutation of Lys145 abolished Rab11a ubiquitylation by β2AR and HACE1 co-expression (Fig. 5A, top panel, lane 11), whereas ubiquitylation of wild-type Rab11a was greatly promoted in the same condition (lane 7). This was reflected in the migration pattern of HA-Rab11-K145R, which showed no ∼33 kDa band in contrast to wild-type HA-Rab11a (Fig. 5A, panels 3 and 5, from top to bottom).
Ubiquitylation of Rab11a on Lys145 is involved in the regulation of β2AR recycling. (A) HEK293 cells were cotransfected with the indicated combinations of FLAG-β2AR, Myc-HACE1, HA-Rab11a and HA-Rab11a-K145R. Immunoprecipitation (IP) of the GTPase was performed with an anti-HA monoclonal antibody. Immunoblotting (IB) was carried out with anti-FLAG, anti-ubiquitin and anti-HA specific polyclonal antibodies. Molecular masses are indicated on the left. The blots shown are representative of three separate experiments. (B–D) HEK293 cells were cotransfected with FLAG-β2AR and the indicated constructs. Cells were treated with 5 µM isoproterenol for 15 minutes at 37°C, and then incubated in DMEM containing 10 µM propranolol for the indicated time periods to prevent further internalization and to allow receptor recycling. Expression of cell-surface receptors was detected by ELISA and the percentage of receptor recycling was calculated. Results are means ± s.e.m. of five separate experiments. Statistical analyses were performed using the regular two-way ANOVA test followed by Bonferroni post-tests. *P<0.05, **P<0.01, ***P<0.001.
The functional role of Rab11a ubiquitylation was then assessed in β2AR recycling experiments in cells that were co-expressing Rab11a, Rab11a-K145R or Rab11a-S25N, alone or together with HACE1 (Fig. 5B–D). As shown in Fig. 5B, co-expression of HACE1 with Rab11a strongly promoted recycling of the receptor compared with when Rab11a was expressed alone 15 minutes after agonist removal (Fig. 5B). However, this effect was not sustained over time. On the contrary, Rab11a-K145R co-expression inhibited the early effect of HACE1 in recycling of the receptor, but also decreased receptor recycling over time (Fig. 5C), to a similar extent as the dominant-negative Rab11a-S25N mutant (Fig. 5D). It is not clear why Rab11a promotes HACE1-mediated recycling of the receptor only 15 minutes after agonist removal. However, it is interesting to note that lack of ubiquitylation of Rab11 on Lys145 seems to confer dominant-negative properties analogous to the well-characterized dominant-negative Rab11a-S25N mutant for the recycling of the β2AR. To verify whether Rab11a-K145R could affect the recycling of another membrane protein, we studied recycling of the transferrin receptor in the presence of co-expressed Rab11a, Rab11a-K145R or Rab11a-S25N (supplementary material Fig. S2). Rab11a-K145R significantly inhibited recycling of the transferrin receptor compared with cells transfected with pcDNA3 or Rab11a, to a degree similar as the Rab11a-S25N mutant. This suggests that Rab11a-K145R regulates the recycling of other membrane proteins. Testing of other receptors will be necessary to conclude whether ubiquitylation of Rab11a is generally involved in recycling of membrane proteins going through that route. It also remains to be determined whether an interaction between other receptors such as the transferrin receptor and HACE1 is involved in the regulation of their recycling by Rab11.
We then wanted to assess the effect of HACE1-mediated ubiquitylation on Rab11a activation. Cells co-expressing HA-Rab11a, -Rab11a-K145R or -Rab11a-S25N with the FLAG-β2AR, alone or in combination with HACE1-Myc, were subjected to fractionation into cytosolic and membrane fractions. An antibody that specifically recognizes Rab11a in its activated, GTP-bound form was used to immunoprecipitate the GTPase and samples were analyzed by western blot using an anti-HA antibody. Fig. 6 shows that HACE1 co-expression promoted Rab11a activation more than twofold but had no significant effect on activation of Rab11a-K145R or of the dominant-negative Rab11a-S25N mutant. Two forms of Rab11a were detected in the cytosolic fractions corresponding to prenylated and unprenylated forms of the protein, whereas only the prenylated form is seen in the membrane fractions (Lachance et al., 2011). These results indicate that ubiquitylation of Rab11a on Lys145 by HACE1 is involved in activation of the small GTPase.
Ubiquitylation of Rab11a on Lys145 by β2AR-HACE1 is involved in its activation. HEK293 cells were transfected with HA-Rab11a, HA-Rab11a-K145R or HA-Rab11a-S25N in combination with FLAG-β2AR alone or with HACE1-Myc. Cells were subjected to fractionation 48 hours after transfection. Cytosolic (C) or membrane (M) fractions were immunoprecipitated with an antibody specifically recognizing activated Rab11-GTP. Immunoprecipitates and samples from the cytoplasmic and membrane fractions were analyzed by western blot using the indicated antibodies. The blots shown are representative of four separate experiments. Densitometry analyses of the quantity of Rab11-GTP that were immunoprecipitated from the membrane fractions were performed with ImageJ software. Densitometry value measured for the β2AR condition was set at 100%. Results are the means ± s.e.m. of four independent experiments. The statistical significance was determined using an unpaired Student's t-test. *P<0.05.
Rab6a and Rab8a are also ubiquitylated by co-expression of β2AR and HACE1
Whether other Rab GTPases were ubiquitylated was then investigated. First, sequence alignments were made between Rab1a, Rab2a, Rab4a, Rab5a, Rab6a, Rab8a, Rab9a, Rab11a and Rac1. Rac1 was included because it was shown to be ubiquitylated by HACE1 on Lys147 (Castillo-Lluva et al., 2012). Interestingly, this analysis revealed that Rab1a, Rab6a and Rab8a have Lys residues located in the vicinity of Rab11a-Lys145 and Rac1-Lys147 between the conserved G4 and G5 boxes (Fig. 7A). The migration pattern of these Rab GTPases was then assessed as for Rab11a in the presence or absence of HACE1 co-expression alone or with β2AR (Fig. 7B). Prolonged exposure of western blot membranes of cell extracts expressing the indicated combinations of proteins showed that higher molecular weight forms were detected in addition to the expected ∼25 kDa proteins for Rab2a, Rab6a and Rab8a, similar to Rab11a (Fig. 7B), suggesting that they could be ubiquitylated. A single band of higher molecular weight can be seen for Rab5 owing to the presence of three HA tags at the N-terminus of the protein. Immunoprecipitation of Rab GTPases that were co-expressed with HACE1 alone or together with β2AR and analyzed by western blotting with a ubiquitin antibody showed that ubiquitylation of Rab6a and Rab8a was detected in basal conditions and enhanced by co-expression of β2AR and HACE1 (Fig. 7C). However, Rab2a ubiquitylation was not noticed. This could be explained by a limitation in sensitivity of the assay or by the fact that the higher molecular weight form detected for Rab2 in Fig. 7B, which was not significantly affected by HACE1 and β2AR co-expression, corresponds to a post-translational modification other than ubiquitylation. Also worthy of note, as in Fig. 5A (second panel), co-immunoprecipitation of HACE1 with the Rab GTPases is augmented by the co-expression of the β2AR in the absence of agonist stimulation (Fig. 7C, second panel), suggesting that the receptor acts as a scaffold in the HACE1 interaction with, and ubiquitylation of, the Rab GTPases.
Identification of Rab6a and Rab8a as HACE1 substrates. (A) Schematic representation of conserved domains of small GTPases and sequence alignments of Homo sapiens Rac1, Rab1a, Rab2a, Rab4a, Rab5a, Rab6a, Rab8a, Rab9a and Rab11a proteins are shown. Conserved residues between G4 and G5 boxes are shown in red and HACE1 ubiquitylation sites of Rac1 and Rab11a are highlighted in yellow. (B) HEK293 cells were cotransfected with FLAG-β2AR and the indicated combinations of pcDNA3, Myc-HACE1 and HA-tagged Rab GTPases. Immunoblotting (IB) was carried out with anti-FLAG, anti-Myc, anti-HA and anti-GAPDH antibodies. The blots shown are representative of three separate experiments. Arrow indicates the appearance of higher molecular mass forms of the Rab proteins upon β2AR and HACE1 coexpression. (C) HEK293 cells were cotransfected with FLAG-β2AR and the indicated combinations of pcDNA3, Myc-HACE1 and HA-tagged Rab GTPases. Immunoprecipitation (IP) of the GTPases was performed with an anti-HA monoclonal antibody. Immunoblotting (IB) was carried out with anti-FLAG, anti-ubiquitin, anti-Myc and anti-HA polyclonal antibodies. Molecular masses are indicated on the left. The blots shown are representative of three separate experiments. (D) Images were prepared with PyMOL (www.pymol.org) using coordinates from crystal structures of active Rac1 (PDB 3TH5), Rab6a (PDB 4DKX), Rab11a (PDB 1OIW) and Rab8a (PDB 3TNF). Potential and confirmed HACE1 ubiquitylation sites are shown in red.
Finally, a comparison of the localization of confirmed or potential ubiquitylation sites on Rac1 (Lys147), Rab6a (Lys144 and Lys146), Rab8 (Lys133, Lys138 and Lys146), as well as Rab11a (Lys145) on the crystal structure of the corresponding proteins is shown in Fig. 7D. This illustrates that these Lys residues are found on the α-helix positioned between the G4 and G5 boxes of the small GTPases and are apparently accessible, as demonstrated for Rac1 (Castillo-Lluva et al., 2012) and Rab11a, for HACE1-mediated ubiquitylation.
DISCUSSION
Cargo-specific regulation of vesicular trafficking has recently attracted much interest. Rab-mediated vesicular transport is well known to regulate membrane receptor trafficking, but less is known about whether membrane receptors conversely regulate the Rab trafficking machinery. We and others showed that GPCRs interact with Rab proteins resulting in the regulation of receptor trafficking (Esseltine et al., 2011; Hamelin et al., 2005; O'Keeffe et al., 2008; Parent et al., 2009; Ritter and Hall, 2009; Seachrist et al., 2002; Smythe, 2002; Wikström et al., 2008). Whether membrane receptors interact with other elements of the Rab-associated machinery to direct their own trafficking is the subject of current intense research. For the important pharmacological GPCR family, unravelling new mechanisms of their trafficking is central to improve our understanding of their regulation and to identify novel possible pharmacological targets. Similarly, the more than 60 members of the Rab GTPase family are involved in virtually every step of vesicular trafficking and are well-documented as targets in human disease. Surprisingly, very little is known regarding their regulation and their interacting partners when considering their number and physiological or pathological importance. The fact that so few Rab GEFs and Rab GAPs have been identified so far is intriguing and could be an indication that other regulatory mechanisms are involved.
In the present study, we showed that HACE1 interacts with the β2AR to regulate its recycling through mechanisms dependent on its E3 ubiquitin ligase activity and Rab11a. HACE1 co-expression reduced the β2AR–Rab11a interaction. This suggested that HACE1 caused Rab11a activation because we previously reported that β2AR interacts with inactive Rab11a (Parent et al., 2009). Mass spectrometry and site-directed mutagenesis showed that Rab11 is ubiquitylated on Lys145 by HACE1. This ubiquitylation is involved in Rab11a activation and in the regulation of β2AR recycling. This is akin to Ras activation by its ubiquitylation on Lys147 (Sasaki et al., 2011). Our data suggest that Rab11 can dimerize. Several GTPases including Ras, Rho and Arf form dimers and oligomers (Beck et al., 2008; Inouye et al., 2000; Zhang et al., 2001; Zhang and Zheng, 1998), although the physiological significance of GTPase oligomerization is not fully understood. Crystallography studies proposed that Rab11a exists as a dimer in its GDP-bound form (Pasqualato et al., 2004). Interestingly, our data indicate that the β2AR, which interacts with the GDP-bound form of Rab11, increased the formation of Rab11a dimers in cellulo, as evidenced by the appearance of a ∼50 kDa band of the predicted size of a Rab11a dimer in cell lysates (Fig. 4A) and by increased co-immunoprecipitation between differentially epitope-tagged Rab11a in the presence of the receptor (Fig. 4B). This was inhibited by co-expression of HACE1. The HACE1–Rab11a interaction was promoted by β2AR co-expression. Altogether, our data suggest that the β2AR acts as a scaffold to promote the HACE1 interaction with, ubiquitylation of and activation of Rab11a. This could possibly result in dissociation of Rab11a dimers from the receptors and interaction of active Rab11a with effectors to regulate β2AR recycling (Fig. 8). However, we cannot exclude the possibility that the receptor promotes the interaction between Rab11a and another ∼25 kDa protein, leading to the appearance of a ∼50 kDa band in cell lysates, or alternatively, that increased co-immunoprecipitation of differentially tagged Rab11a is caused by the formation of receptor dimers or oligomers, and that both of these processes would be reduced by the expression of HACE1. More work will be needed to fully address whether Rab11a dimerizes.
Model for Rab11a activation by β2AR through HACE1-mediated ubiquitylation. Briefly, β2AR interacts with Rab11a dimers (question mark indicates that Rab11a dimerization is a possibility that remains to be confirmed) and acts as a scaffold to recruit HACE1 (step 1) resulting in the ubiquitylation of Rab11a (step 2), leading to Rab11a GTP-loading and dimer dissociation (step 3), and ultimately to dissociation from the receptor and activation of effectors to regulate β2AR recycling (step 4).
Rab4a was not ubiquitylated by β2AR–HACE1, correlating with its inability to enhance HACE1-mediated β2AR recycling. Rab6a and Rab8a, but not other Rab proteins tested in the present study, were ubiquitylated in the presence of β2AR and HACE1. It will be interesting to further characterize this specificity towards other Rab proteins and to determine whether other GPCRs exhibiting different trafficking itineraries behave like the β2AR. HACE1 has previously been shown to interact with Rab proteins, but no effect on Rab ubiquitylation was described (Tang et al., 2011). This could be due to the absence of a crucial factor in the equation, similar to for example the co-expression of a GPCR. Future studies will reveal whether this is specific to this class of membrane receptors but our data with the transferrin receptor suggest that ubiquitylation of Rab11 is involved in the regulation of recycling of other receptor types. It will be interesting to determine the role of HACE1 in regulating trafficking of other membrane receptors through Rab11, and whether other receptor types modulate ubiquitylation of Rab GTPases. Ubiquitylation of Rac1 on Lys147 by HACE1 causes proteasomal degradation of the small GTPase (Castillo-Lluva et al., 2012). The stability of Rab6a, Rab8a and Rab11a was not affected by HACE1-mediated ubiquitylation, suggesting that mono-ubiquitylation or ubiquitin chains incompatible with proteasome-mediated degradation are involved. Specificity thus seems to also exist in the effects of ubiquitylation of target proteins by HACE1. Specificity was also shown by the inability of Nedd4 to induce shifts in the migrating pattern, reflecting ubiquitylation of Rab11a, in contrast to results obtained with HACE1.
How ubiquitylation of Rab11a leads to its activation remains to be determined. Mono-ubiquitylation of Ras on Lys147 severely abrogates its interaction with and the response to GAPs (Baker et al., 2013). However, some GEFs for other small GTPases, for instance Rabex 5 for Ras, c-Cbl for Rap1 and Rac1, and HERC1 for Arf1, Rab3a and Rab5, were shown to be E3 ubiquitin ligases (Barr and Lambright, 2010; Rosa et al., 1996; Swaminathan and Tsygankov, 2006; Xu et al., 2010). Whether HACE1 acts as a GEF for Rab11a or whether ubiquitylation of Rab11a modulates its interaction with GEFs or GAPs will be the subject of future experiments. Rab11 GEFs are known in Drosophila (Xiong et al., 2012) but no human orthologs have yet been identified to the best of our knowledge.
It is also noteworthy that β2AR-HACE1-mediated ubiquitylation shows specificity for Rab6a, Rab8a and Rab11a. This is analogous to our recent discovery of a complex between the β2AR and Rab geranylgeranyltransferase α that regulates the prenylation of these three GTPases (Lachance et al., 2011). This is interesting because Rab6a, Rab8a and Rab11a are functionally connected in intracellular transport: Rab11 interacts with Rabin8 and stimulates its GEF activity toward Rab8 (Knödler et al., 2010) and Rab6a-interacting protein 1 links Rab6 and Rab11 function (Miserey-Lenkei et al., 2007). In vitro studies indicate that Rab6 does not interact with HACE1 (Tang et al., 2011). However, we observed that HACE1 mediates Rab6a ubiquitylation. This could be explained by the lack of post-translational modifications of the purified proteins that prevent them from interacting in vitro. This is however unlikely because HACE1 directly interacts with Rab11 in the same conditions (Tang et al., 2011). It is thus possible that an intermediate is involved in the interaction between HACE1 and Rab6 to mediate the ubiquitylation of this small GTPase. Perhaps Rab6a could get ubiquitylated by HACE1 as part of a functional complex with Rab11a that remains to be defined.
The fact that HACE1-C876S was able to promote cell surface expression of β2AR suggests that the protein has functions that are independent of its E3 ligase activity. HACE1 contains six putative ankyrin repeat domains usually known for their scaffolding properties in protein complex assembly (Mosavi et al., 2004). We recently showed that ankyrin-repeat-containing proteins can promote export of GPCRs (Parent et al., 2010). It is thus possible that HACE1 increases cell surface targeting of the β2AR through protein interactions with its ankyrin repeats.
In summary, our findings provide significant novel insights into three issues of intracellular trafficking and regulation of Rab GTPases: (1) Rab GTPases can be ubiquitylated; (2) ubiquitylation of Rab11a is involved in its activation; and (3) cargo proteins such as GPCRs, can regulate their own trafficking by regulating the activity of Rab GTPases through scaffolding complexes between the Rab GTPases and an E3 ubiquitin ligase. This could be relevant to human diseases associated with impaired GPCR trafficking and dysregulation of Rab GTPases.
MATERIALS AND METHODS
Reagents
The monoclonal anti-HA (16B12) and anti-Myc (9E10) antibodies were from Covance. The polyclonal anti-FLAG, monoclonal FLAGM1-specific and the polyclonal anti-HACE1 were from Sigma-Aldrich. The mouse monoclonal IgG1 FLAGM2 antibody was also used as an isotypic control. The polyclonal anti-Myc (A-14), the anti-HA-probe (Y-11), the anti-GAPDH antibodies and Protein-G agarose beads were from Santa Cruz Biotechnology. The monoclonal anti-HA-peroxidase (3F10) was purchased from Roche Applied Science. The mouse monoclonal anti-Rab11a was purchased from BD Transduction Laboratories. Anti-mono- and anti-polyubiquitinylated monoclonal antibodies conjugated to peroxidase (FK2H) were from Enzo Life Sciences. The anti-Myc-HRP polyclonal antibody was from Abcam. The anti-active Rab11 antibody was from NewEast Bioscience. The rabbit monoclonal anti-Rab11a, Alexa Fluor 546 donkey anti-rabbit, Alexa Fluor 633 goat anti-mouse antibodies, human transferrin conjugates to Alexa Fluor 546, and ProLong Gold antifade reagent were purchased from Invitrogen. Isoproterenol, propranolol and monensin were purchased from Sigma. An alkaline-phosphatase-conjugated goat anti-mouse antibody and the alkaline phosphatase substrate kit were purchased from Sigma. Human holo-Transferrin was a kind gift from the laboratory of Dr Richard Leduc (Université de Sherbrooke).
Plasmid constructs
The HA-Rab11a-S25N, HA-Rab11a-K145R constructs were prepared by PCR from the pcDNA3-HA-Rab11a constructs as described previously (Thériault et al., 2004; Parent et al., 1999). The HA-Nedd4 construct was purchased from Addgene (plasmid 11426). The Myc-HACE1 construct was prepared by PCR from the human cDNA clone template purchased from OriGene (SC107534). The PCR fragment was digested with BamHI and EcoRI and ligated into the pcDNA3 vector. The Myc-HACE1-C876S mutant was prepared by PCR from pcDNA3-Myc-HACE1. The full-length PCR fragment was digested with BamHI and EcoRI and ligated into the pcDNA3 vector. Integrity of the coding sequence of these constructs was confirmed by dideoxy sequencing.
Cell culture and transfection
Human embryonic kidney HEK293 cells were maintained in DMEM (Dulbecco's Modified Eagle's Medium) (Invitrogen) supplemented with 10% (v/v) FBS (fetal bovine serum) at 37°C in a humidified atmosphere containing 5% CO2. Transient transfection of HEK293 cells grown to 50–70% confluence were performed using the TransIT-LT1 Reagent (Mirus) according to the manufacturer's instructions. Empty pcDNA3 vector was added to keep the total amount of DNA per plate constant.
Immunoprecipitation
HEK293 cells were transiently transfected with the indicated constructs and were maintained as described above for 48 hours. The cells were then washed with ice-cold PBS and harvested in 300 µl of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.5% deoxycholate, 0.1% SDS, 10 mM Na4P2O7, 1% IGEPAL, and 5 mM EDTA or 1 mM CaCl2 depending on the antibody used for the assay) or ubiquitylation lysis buffer for ubiquitylation experiments (50 mM HEPES, pH 7.5, 250 mM NaCl, 2 mM EDTA or 1 mM CaCl2, 0.5% IGEPAL, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, 10% glycerol and 10 mM N-ethylmaleimide). Both buffers were supplemented with protease inhibitors (9 nM pepstatin, 9 nM antipain, 10 nM leupeptin and 10 nM chymostatin) (Sigma Aldrich). Immunoprecipitations were then carried out as we described previously (Lachance et al., 2011; Parent et al., 2010).
Immunofluorescence staining and confocal microscopy
Confocal microscopy was performed to detect the indicated endogenous or transfected proteins in HEK293 cells using a scanning confocal microscope (FV1000-Olympus) coupled to an inverted microscope with a ×60 oil-immersion objective lens and images were processed using Fluoviewer 2.0 software (Olympus). Cells were processed as we described previously (Lachance et al., 2011; Parent et al., 2010).
Measurement of cell-surface receptors
For quantification of expression of cell-surface receptors, 6.5×105 HEK293 cells were plated in 24-well plates pre-coated with 0.1 mg/ml poly-L-lysine (Sigma), transfected with the indicated constructs and then maintained for an additional 48 hours and then processed for ELISA as described previously (Hamelin et al., 2005; Lachance et al., 2011; Parent et al., 2009; Parent et al., 2010; Parent et al., 1999; Thériault et al., 2004).
siRNA assays
The synthetic oligonucleotides ID s33239 and s33240 targeting the human HACE1 gene, s16703 and s16704 targeting the human RAB11A gene, and the negative control siRNA (Silencer Negative Control 1, catalogue number-AM4611) were purchased from Ambion. HEK293 cells were transfected with 10 nM oligonucleotide using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Protein expression analysis by western blotting and ELISA experiments were performed at 72 hours post-transfection as usual (Lachance et al., 2011; Parent et al., 2010).
DMP antibody crosslinking
For HA-Rab11a purification needed for mass spectrometry experiments, we generated Protein-G agarose beads DMP (New England Biolabs) crosslinked to mouse IgG1 anti-HA antibody as described by the manufacturer. Briefly, 100 µl of Protein-G agarose beads were resuspended and aliquoted in test tubes. Beads were washed twice with 500 µl of binding buffer (0.1 M sodium phosphate buffer pH 8.0). Then 80 µl of binding buffer and 30 µg of anti-HA antibody were added and incubated overnight on a rocker at 4°C. The following day, beads were washed three times with 500 µl of binding buffer and twice with 1 ml of crosslinking buffer (0.2 M triethanolamine, pH 8.2). Subsequently, 1 ml of fresh DMP solution (15 mM in crosslinking buffer) was added and crosslinking was allowed for 60 minutes on a rocker at room temperature. Beads were then washed once with blocking buffer (0.1 M ethanolamine, pH 8.2) and incubated in 1 ml of blocking buffer for 60 minutes on a rocker at room temperature. Beads were washed once with PBS then unbounded antibodies were eluted twice with 1 ml of elution buffer (0.1 M glycine pH 2.5). Finally, beads were washed twice with PBS, resuspended in 50 µl PBS, and stored at 4°C until use.
Protein purification and direct LC-MS/MS analysis
HEK293 cells were plated in five 100 mm Petri dishes at a density of 2.0×106 cells per dish. The following day, the cells were transiently transfected with FLAG-β2AR, HA-Rab11a with or without Myc-HACE1 and then maintained for an additional 48 hours. The cells were then treated with 0.75 µM of epoxomicin or 0.42% DMSO for 4 hours. Subsequently, the cells were washed twice with ice-cold PBS and harvested in 1 ml per dish of ubiquitylation lysis buffer supplemented with protease inhibitors. After 60 minutes of incubation in lysis buffer at 4°C, the lysates were centrifuged for 30 minutes at 13,500 g at 4°C. Lysates were pooled and pre-cleared with 20 µl of Protein-G agarose beads per ml of lysate for 1 h on a rocker at 4°C and then pre-cleared again with 20 µl of Protein-G agarose beads per ml overnight. The following day, 100 µl of Protein-G agarose beads DMP-crosslinked to mouse IgG1 anti-HA antibody was added to pre-cleared lysates and incubated overnight on a rocker at 4°C. Beads were then washed three times with lysis buffer, and three times with 50 mM ammonium bicarbonate, pH 7.8, to remove any residual detergent. Finally, the antibody complex was eluted four times with 100 µl of elution buffer (0.1 M glycine, pH 2.5), pre-heated to 37°C, for 10 minutes on a rocker at 30°C. The eluted fractions were neutralized with 10 µl of Tris-HCl, pH 8.0. The resulting peptide mixture was analyzed by liquid chromatography-tandem MS (LC-MS/MS) using a LTQ-XL Linear Ion Trap Mass spectrometer (Thermo Scientific) as described previously (Daulat et al., 2012).
Subcellular fractionation
HEK293 cells were grown overnight in 100 mm Petri dishes before being transfected. Forty-eight hours after transfection, cells were suspended in 1 ml of hypotonic buffer (0.1× PBS) supplemented with protease inhibitors (9 mM pepstatin, 9 mM antipain, 10 mM leupeptin and 10 mM chymostatin) and incubated on ice for 10 minutes before being broken with 20–30 strokes of a Dounce homogenizer depending on cell confluency. 100 µl of 10× PBS was then added to the hypotonic cell solution to obtain a 1× concentration. Cells were then centrifuged at 3000 rpm for 20 minutes at 4°C. Supernatant was conserved and was centrifugated again at 10,000 rpm for 10 minutes at 4°C to remove nuclei, unlysed cells and large cell debris. Lysates were then centrifuged at 100,000 g for 1 hour at 4°C to give supernatant and pellet fractions. The fractions were subjected to immunoprecipitation and western blot analysis using specific antibodies.
Densitometry analyses
The densitometry analyses were performed with ImageJ software.
Statistical analysis
Statistical analyses were performed using Prism version 5.0 (GraphPad Software) using the unpaired or paired Student's t-test and Two-way ANOVA test followed by Bonferroni post-tests. Data were considered significant when *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Footnotes
Competing interests
The authors declare no competing interests.
Author contributions
V.L., J.D., S.M., M.R., S.G. and S.N. performed the experiments. V.L., S.A. and J.L.P. conceived the experiments, analyzed the data and wrote the manuscript.
Funding
This work was supported by the Canadian Institutes of Health [grant number MOP-184095]; and a Chercheur Senior salary award from the Fonds de la Recherche Québec-Santé (FRQS) to J.L.P. J.L.P. is the recipient of the André-Lussier Research Chair. V.L. received a studentship from the FRQS during part of this work.
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.132944/-/DC1
- Received April 12, 2013.
- Accepted October 12, 2013.
- © 2014. Published by The Company of Biologists Ltd
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