Association of Connexin43 with E3 Ubiquitin Ligase TRIM21 Reveals

Article pubs.acs.org/jpr

Association of Connexin43 with E3 Ubiquitin Ligase TRIM21 Reveals a Mechanism for Gap Junction Phosphodegron Control Vincent C. Chen,†,‡ Anders R. Kristensen,† Leonard J. Foster,*,†,# and Christian C. Naus*,‡,# †

Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, ‡Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3, Canada S Supporting Information *

ABSTRACT: Gap junctions (GJs) are sites of direct cell-tocell communication formed by the connexin (Cx) family of ion channel proteins. The aberrant intercellular communication mediated by GJs is associated with a variety of hereditary and acquired human diseases. GJs utilize a highly interconnected network that is indispensible for synthesis, trafficking and degradation of their constituent proteins. By unbiased proteomic examination and network enrichment, we identified interacting components of the ubiquitin proteasome system associated with Cx43. LC-MS/MS identification and quantification of tryptic peptides from IP materials revealed a variety of interacting candidates, including the E3 ligase TRIM21 and ubiquitin. The interaction of Cx43 with TRIM21 was confirmed by confocal microscopy and coimmunoprecipitation of these proteins from C6 rat glioma and mouse primary astrocyte cultures. To gain a better understanding of this interaction, complexes isolated by high-resolution size-exclusion chromatography revealed signal integration by phosphorylation, ubiquitylation and proteolytic turnover within complexes of Cx43/TRIM21. Cx43/TRIM21 is also responsive to E1 UBE1 and E2 UbcH5a, with the interruption of this activity being an effective inhibitor of in vitro ubiquitin-conjugation. Mathematical models of these complexes demonstrated a mechanism for the switch-like degradation of GJs that were validated in EGF-stimulated cell cultures. Our finding of the interaction of Cx43 with TRIM21 provides mechanisms for the down-regulation of GJ intercellular communication that are known to impact a variety of physiological processes. KEYWORDS: Cx43, TRIM21, phosphorylation, ubiquitin, E3 ligase, gap junction, phosphodegron

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INTRODUCTION Gap junctions (GJs) are sites of intercellular communication located within the plasma membranes of adjacent cells that permit the exchange of small ions and metabolites (generally 2 MDa) were not commercially available, column void volume (Vo) was determined by separating whole cell lysates under native conditions with Vo 6136

dx.doi.org/10.1021/pr300790h | J. Proteome Res. 2012, 11, 6134−6146

Journal of Proteome Research

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Figure 2. Network analysis. (A) Global network of screened and preexisting Cx43-protein interactions and molecular function analysis. Signaling kinases, including EGFR, SRC, CDK1, ERK1/2 and P38MAPK, are identified in red. Proteins identified with indicated cellular processes (CP). (B) Table of statistically enriched canonical pathways of Cx43 interaction candidates. (C) A proposed mechanism of ubiquitylation and degradation mediated by RING E3 ubiquitin ligase in response to protein phosphorylation.

represented by the first appearance of protein as monitored by UV. To calibrate complex size as a function of elution volume/ time, a standard protein mixture was separated (Sigma, MW-GF1000). As expected, elution volume (Ve) was observed to decrease linearly with the logarithm of the molecular weight, with R2 values of 0.95 or better. Collected samples were then precipitated with aqueous TCA (100% w/w), centrifuged, rinsed with acetone and air-dried for suspension in denaturing SDSPAGE buffer for analysis by SDS-PAGE/Western blotting. Protein intensities were quantified with GelEval 1.22 (Frogdance Software) and normalized to maximum intensities within each series. Normalized values for Cx43, TRIM21, and their posttranslational modified variants were transferred to Excel and SigmaPlot for presentation and mathematical modeling.

incubated with covalently conjugated agarose beads (Amino-link Kit, Pierce) corresponding to 3 μg of anti-TRIM21, or anti-Cx43. Incubations were performed with rotation for 1 h at 4 °C. For in vitro ubiquitylation assays, TRIM21 IPs (3 μg of rabbit polyclonal anti-TRIM21 Zymed/Invitrogen) and their respective controls were exposed to lysates of C6-Cx43 cells or mouse astrocytes. For in vitro ubiquitylation, IPs were taken through the same procedure except incubations were conducted at room temperature for 30 min or 3 h intervals, overnight prior to isolation and wash. Four 10 min washes were conducted using 1 mL per IP wash cycle (containing inhibitors for protease and phosphatase). Samples for IP negative controls were taken through the same procedure except for the addition of preimmune mouse/rabbit/ goat IgG. Samples were then denatured in SDS-PAGE loading buffer prior to separation on a 4−20% gradient gel. For reconstituted ubiquitylation assays, TRIM21 IPs were incubated using a ubiquitin conjugation reaction kit, 26S proteasome, ubiquitin, UBE1 and Ubc5Ha (Boston Biochemical, Cambridge, MA).

Immunoprecipitation and TRIM21 Ubiquitylation Assays

C6-Cx43 cells were grown to 90% confluence in three 10 cm dishes. Cells were lysed in 1 mL of IP buffer, scraped and briefly sonicated. Cellular debris was removed by centrifugation at 17 500 rcf for 5 min and divided for IP and negative (750 μL of corresponding to 2 mg protein). For IPs, supernatants were 6137



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Figure 3. Association, colocalization and in vitro ubiquitylation of Cx43/TRIM21. (A) Co-purification of K48-sites of ubiquitylation within Cx43 co-IPs by LC-MS/MS. Residues carrying the dimethylation are indicated (*). (B) Reconstructed chromatograms demonstrate the isolation of the K48-glygly tryptic peptide (black line) and the lack of this material in IP negative control (red). (C) TRIM21 IP blotted for Cx43 (lane 3) shows the association of diubiquitylated protein. IPs of endogenous ubiquitin show the positions of Cx43 and mono- and diubiquitylates at respective 40 and 50 kDa bands (lane 7). Whole cell lysates (lanes 1 and 4) with IPs from normal IgG (Lanes 2 and 5) serve as respective controls for positive and negative detections. (D) Kinetic assessment of ubiquitylation from IPs of TRIM21. At 30 min Cx43 exists as diubiquitylated protein (lanes 3 and 8), and during extended incubation (3 h), higher MW forms of Cx43 are observed (lanes 5 and 11). Serving as negative controls, Cx43 was not detected with normal IgG lanes (2, 4, 7 and 10). (E) Laser scanning confocal images of TRIM21 and Cx43 labeled in C6-Cx43 cells. Cx43 with TRIM21 together (yellow) within merged and inset merged images demonstrate labeling at cell-to-cell contacts (white arrows) and intracellular pools. Blue: DAPI nuclear staining. Scale bar: 10 μm.

Immunofluorescence Microscopy

(Prolong, Invitrogen). Co-localization images were acquired using a 60X Plan S-Apo (1.42N.A. − oil) objective lens on a confocal (Fluorview 1000 IX81, Olympus) microscope with all images acquired in sequential scan mode at 36.7 °C. Images in Fluoview software were exported as TIFF files from Macification (v.1.5.2, Obicule), assembled in Photoshop Elements 6.0 (Adobe) and presented as unaltered images. Mander’s correlation coefficients for Cx43/TRIM21 were obtained using the JACoP plug-in for ImageJ.

C6-Cx43 cells were cultured to confluence on glass coverslips and rinsed with PBS (with Mg2+, Ca2+). Cells were fixed with 10% phosphate buffered formalin for 5 min (RT) and blocked with 2% BSA in PBST (PBS pH 7.4 containing 0.3% Triton X100) for 30 min (RT). Primary antibodies were diluted in 1% BSA PBST and applied onto coverslips. Cells were then washed in PBS for 3 × 10 min and in 1% BSA PBST, and treated with secondary probes in 1% BSA PBST for 1 h at room temperature in the dark. Coverslips were then washed for 1 h in PBS and mounted on glass slides with antifade medium containing DAPI 6138



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RESULTS

demonstrated 50−55 kDa bands consistent with the stoichiometric addition of 2 ubiquitins (Figure 3C, lane 7). As only a small fraction of Cx43 was found to be ubiquitylated within antiubiquitin co-IPs (estimated to be less than 1% by densitometry), this observation suggests Cx43 ubiquitylates largely reside with an overwhelming abundance of nonubiquitylated connexin, likely as noncovalent GJ assemblies that are actively undergoing turnover. In contrast to nonubiquitylated Cx43 principally found within positive control whole cell lysates (lane 1), co-IPs of TRIM21 interestingly demonstrated reactive higher MW bands, which were consistent with Cx43-ubiquitin post-translational modification and functional association with a protein having E3 ligase activity (lane 3). This observation would suggest ubiquitylates of Cx43 were specifically coprecipitated with TRIM21, or possibly, coisolation and activity of TRIM21 resulted in Cx43 ubiquitylation and loss of Cx43 at parent mass (∼37−45 KDa). Demonstrating this scenario, and guiding our experiments (Figure 3D), we also noted within earlier examinations that the MW of Cx43 ubiquitylates appeared to positively correlate with the time IPs were exposed to whole cell lysates. Indeed, to confirm E3 ligase activity toward Cx43 under controlled in vitro conditions, TRIM21 IPs were subjected to in vitro reconstitution assays to monitor levels of Cx43 ubiquitylation by SDS-PAGE Western blot. Similar to the classic use of rabbit reticulocyte lysates,57 fresh preparations of C6-Cx43 cells and astrocytes were used to examine rates of ubiquitylation. In the presence of broadspectrum protease inhibitors, mono-/diubiquitylated Cx43 started accumulating by 30 min (Figure 3D, lanes 3 and 8) and by 3 h most Cx43 had been populated by poly-/multiubiquitylates in both C6-Cx43 and astrocyte materials (Figure 3D, lanes 5 and 11). As important indicators that reactions were specific to TRIM21 (Figure 3D, lanes 3, 5, 8 and 11), no mass shifts of Cx43 were observed with negative controls using bulk lysates from astrocytes (Figure 3D, lanes 6 and 9) or by immunoadsorbing proteins with nonspecific rabbit IgG (lanes 7 and 10).

Cx43-Protein Interaction and Network Analysis

For proteomic examinations, lysates of C6-Cx43 cells expressing endogenous and exogenous Cx4345 and its interacting components were immunoprecipitated (IP) and analyzed by LC-MS/MS (Figure 1A). Under native, nondenaturing conditions, Cx43 complexes were distinguished from nonspecific assemblies by stable isotope dimethyl labeling.47 Peptides of trypsin (added at equal abundance) serve as quantification controls, with identified Cx43 peptides serving as positive IP controls. A total of five biological replicates were completed, which included three LC-MS analyses along with two larger-scale experiments where peptides were prefractionated by pH 10 ammonium formate C18 chromatography (five fractions/ sample). Among those proteins demonstrating enrichment with Cx43 IP (as described within the methods), we identified several previously identified Cx43-protein interactions, such as α/β-tubulin49 and ubiquitin,41,42 as well as a potential interaction with the RING domain E3 ubiquitin ligase TRIM21 (Figure 1B,C). Proteins identified here were observed at high confidence, with quantitative ratios suggesting that these proteins may be capable of associating with Cx43. Within this screen we observed several functional classes: (i) protein ubiquitylation pathway, (ii) integrin signaling, (iii) actin cytoskeleton signaling, (iv) regulation of actin-based motility by Rho, and (v) clathrin mediated endocytosis (Figure 2). An in silico evaluation of these networks versus a same-sized pool of random interactions revealed that the networks identified were significant with Pvalues ranging from 8.85 × 10−7 to 3.87 × 10−5 using the BIND, DIP, MINT, MIPS, BIOGRID, INTACT, IPA Knowledgebase, and COGNIA databases. Quantified proteins (p < 0.05) and peptides identified by this study are provided in Supporting Information (Table S1, S2). Ubiquitin Conjugation and Catalysis of Cx43/TRIM21 Assemblies

The proteins bound specifically to Cx43 suggest that the activities of the ubiquitin-proteolytic system may be responsible for preventing the build up of GJs. In particular, the E3 ubiquitin ligase TRIM21 forms a Skp-Cullin-Fbox/SCF-like complex regulating the ubiquitylation of p27.50−53 In light of the identification of TRIM21 and the ubiquitin proteasome system, we next researched MS fragment spectra, this time allowing for the glygly ubiquitin-tag on lysine. Authenticating ubiquitin identifications, K48-ubiquitin peptides (Figure 3A) were detected with quantitative ratios above those of Cx43 and TRIM21 suggesting assembly within identified complexes (Figure 3B). To help confirm this identification, we further examined digests of purified K48-polyubiquitin chains to yield MS/MS spectra54 encompassing the expected missed sites of lysine/trypsin cleavage55 (data not shown). Although the data in this study demonstrate that both Cx43 and TRIM21 are ubiquitylated, we were unable to identify the direct sites of ubiquitin or phosphate conjugation. This was likely due to the presence of abundant proteins observed within Cx43 IPs that likely masked their detection. In light of our findings thus far, we further sought to examine the signaling mechanisms of ubiquitylation and the interaction of Cx43 with TRIM21. To investigate Cx43/TRIM21 interaction, cell lysates were subjected to reciprocal TRIM21 IP for Cx43 detection (Figure 3C). To validate ubiquitin conjugation, membranes reprobed with antiubiquitin antibodies confirmed ubiquitylated Cx43 bands at 50−55 kDa. Cx43 immunoblots of ubiquitin IPs

Co-trafficking and Assembly of TRIM21/Cx43 Complexes at Gap Junctions

To investigate the functional association of Cx43 with TRIM21, we next examined the colocalization of these proteins by immunofluorescence/laser scanning confocal microscopy. The distribution and colocalization of Cx43 and TRIM21 were examined by double immunofluorescence labeling of these proteins in cultures of C6-Cx43 cells. As shown in Figure 3, the two proteins appear together along strands of heavy labeling at points of intercellular contact and in trafficking bodies, with a subset of these proteins appearing to have been internalized from the plasma membrane (Figure 3, inset). Observed weakly in the cytoplasm, TRIM21 was more prominently found along plasma membranes and other regions where the two proteins were often colocalized. These observations are particularly relevant in the context of connexin maturation, where the protein is known to traverse multiple organelles and cellular domains. Indeed, Manders’ correlation coefficient of 0.80, measuring the colocalization of Cx43 with TRIM21, further imply the proteins undergo a significant level of cotrafficking as a complex. To confirm this was not an artifact of Cx43 overexpression, examinations of Cx43/TRIM21 distributions in wild-type primary astrocytes demonstrated similar Manders’ correlation (0.82) and labeling at GJ structures (Supplemental Figure S1, Supporting Information). With previously demonstrated activity, 6139



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Figure 4. Complex profiling by chromatography. (A) C6-Cx43 complexes were separated by SEC followed by SDS-PAGE (4−20%)/Western blot, with indicated proteins and positions of Cx43 ubiquitylation, phosphorylation (phos), nonphosphorylation (nonphos), proteolytic degradation (deg), TRIM21 and TRIM21 ubiquitylation. (B) SEC molecular weight calibration using indicated protein standards. Analysis of separated complexes reveals increases in Cx43 phosphorylation within complexes of highest mass. Overlay of these events indicates mutual assembly within profiled SEC fractions (lanes 1−6). Plots are normalized to maximum abundance. (C) Brefeldin A treatment of C6-Cx43 cells demonstrates inhibition of phosphorylated Cx43 bands relative to untreated control. GAPDH controls demonstrate equal loading within these lanes. (D) Preparations of enriched GJs demonstrate phosphorylated/ubiquitylated Cx43. Calf intestinal alkaline phosphatase (CIP) treatment results in the collapse of phosphorylated materials.

connexin rich-structures (commonly termed connexosomes). Suggesting the Cx43/TRIM21 platform mediates signaling cross-talk between kinases and ubiquitin ligases, it was interesting for us to note complexes of highly phosphorylated versions of Cx43 also harbored ubiquitylates of both Cx43 and TRIM21. To confirm that levels of post-translational modification were a true reflection of Cx43 at GJs, we blocked ER-to-Golgi trafficking and GJ formation with brefeldin A.57 Inhibiting Cx43 trafficking with this reagent resulted in the near complete loss of phosphorylated and ubiquitylated Cx43 (Figure 4C). As seen in blots loaded with equal protein, treatment with brefeldin A also coincided with an accumulation of N-terminal processed Cx43 (35 kDa) that is likely attributed to increased ER load and signal peptidase cleavage.58 To further confirm this view, we employed an orthogonal method to enrich GJs by differential centrifugation.59,60 Examination of Cx43 by this method demonstrated similar patterns of Cx43 phosphorylation and ubiquitylation consistent with GJ formation and turnover (Figure 4D).

these data are consistent with Cx43/TRIM21 assemblies mediating the ubiquitylation and turnover of Cx43 GJs. Contributions of TRIM21 Association and Post-Translational Modification in the Assembly and Turnover of Cx43/GJs

The connexin life-cycle is comprised of an array of signaling events, including phosphorylation and protein−protein interactions. With aims of elucidating the mechanisms of GJ turnover, and to remove possible biases introduced by coisolation using TRIM21 antibodies, we next sought to examine complexes of Cx43/TRIM21 by high-performance size exclusion chromatography (SEC). Here, complexes within C6-Cx43 cell lysates were isolated using a high-resolution SEC column capable of resolving complexes up to at least 2 MDa (Figure 4A, fractions 1−25) using approaches recently developed by our lab.56 Within these profiles, Cx43 demonstrates quantitative increases in phosphorylation and mass that appeared to reflect the intrinsic stages of the Cx43/GJ lifecycle, namely, biosynthesis, GJ hemichannel, GJ assembly, Cx43 phosphorylation at GJs, and turnover as large 6140



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Treatment of this material with phosphatase and analysis by SDS-PAGE/Western blotting demonstrated the collapse of phosphorylated and ubiquitylated connexin, to their respective nonphosphorylated forms with faster SDS-PAGE migration. Specifically observed within these complexes, Cx43/TRIM21 ubiquitylation, high percentages of Cx43 phosphorylation, alongside bands that are consistent with Cx43 C-terminal cleavage, suggested these events were derived from the same degradation processes. In particular, our findings of ubiquitylated/degraded Cx43 and TRIM21 strictly residing within complexes of highly phosphorylated connexin (45 kDa), and not the reverse, supports the notion of a unidirectional dependence of ubiquitylation and cleavage on Cx43 phosphorylation levels. As initially observed with ubiquitin IPs (Figure 3C, lane 7), this examination orthogonally finds that only a small percentage (150 kDa) by the 26S proteasome could not be fully ruled out, suggesting this scenario was not the case, the intensity of these bands did not scale with increasing proteasome concentrations (lanes 4 and 5). While Cx43 ubiquitylates were found to be largely nonresponsive, demonstrating 26S activity, TRIM21 was found to be massively sensitive to proteolysis and scaled with proteosome addition (516 kDa) and coalesce into sites of GJs (≫516 kDa).64,65 During turnover, GJs can undergo internalization as large double membrane vesicles, called annular GJs23 or connexosomes.33 Plasma membrane Cx43 associates with many proteins, including a variety of kinases that induce massive changes in phosphorylation.33,66 Despite a wealth of information related to its degradation, there is still a great deal of uncertainty over ubiquitylation as a principle mechanism of connexin control. Using a lysine-less mutant, Su et al. recently provided very interesting evidence that Cx43 can still be degraded by the

Figure 6. Phosphodegron modeling. Modeled equation (eq 1) and statistical analysis for equations describing TRIM21 ubuiquitylation as a function of the magnitude of Cx43 phosphorylation. First order derivative (eq 2) provides a threshold value of 83.4% (eq 3) to initiate GJ phosphodegron activity.

sought to validate our model using a system that has been previously found to initiate the loss of GJ intercellular communication with EGF treatment.34,38 Using C6-Cx43 cultures and previously described methods for complex profiling,

Figure 7. Examination of the GJ Phosphodegron with EGF stimulation. C6-Cx43 cells treated with EGF (100 ng, 5 h) were quantitatively profiled by SEC-SDS-PAGE/Western blot. Detections of (A) Cx43 and (B) TRIM21 identify isolated complexes presenting evidence for phosphorylation, ubiquitylation and degradation. (C) EGF stimulated conditions resulted in increased number of complexes presenting high phosphorylation percentages (80−100%) relative to untreated controls. (D) Consistent with an induced phosphodegron previously modeled (Figure 6), EGF-induced phosphorylation expanded the number of GJ assemblies available for ubiquitylation and degradation, as determined by the appearance of modified bands within complexes harboring both Cx43 and TRIM21. 6142



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proteasome and highlighted the fact that the “exact subcellular location(s) of Cx43 ubiquitination are still unknown”.26 Follow up work by Dunn et al. using the same mutant construct further demonstrated proteasomal degradation of GJs did not involve lysine-dependent ubiquitylation.67 The majority of studies investigating the link between Cx43 and channel regulation have historically focused on phosphorylation as the principle mechanisms of GJ post-translational control, turnover and channel closure. Over the course of this study, we found the fundamental difficulty of studying GJ ubiquitylation was the challenge of isolating assemblies actively undergoing degradation. Motivating these efforts, a significant body of evidence suggests that ubiquitin plays an important role in the internalization and endosomal sorting of plasma membrane ion channels and receptors that suggest these events may also be extended to Cx43. The loss of GJ intercellular communication is believed to be an important early step in cancer cell progression and the EGF induced phosphorylation of Cx43 correlates with the decrease in GJ intercellular communication, and the loss of GJs from the cell surface.38,40,41,68−71 In this study, we performed an unbiased examination to identify interacting proteins by LC-MS/MS. This strategy confirmed previously identified Cx43 interactions (α-, βtubulin, and ubiquitin), along with the specific interaction of the E3 ubiquitin ligase TRIM21. The functional interaction of Cx43 with TRIM21 was supported by co-IP of these proteins, colocalization, and high-precision complex profiling by SEC HPLC, along with assays to induce ubiquitylation and the kinetic consumption of phosphorylated Cx43 substrates. Consistent with presented models, conditions stimulating EGF/EGFR signaling pathways in C6-Cx43 cells were found to dramatically increase the number of ubiquitylated complexes presenting Cx43 degradation via proteolysis of the protein’s c-terminal tail. Through our identification of Cx43 association with TRIM21, we provide important mechanistic details outlining the ubiquitylation and turnover of GJs. A phosphodegron is defined as one or a series of phosphorylated residues in a protein that promotes its own turnover  usually in collaboration with acting E3 ubiquitin ligases.72−75 Our finding that Cx43 phosphorylation triggers its ubiquitylation and turnover is consistent with previous reports showing individual sites of phosphorylation, including Y247,76 S255,34 S262,77 Y265,76 S279 and S282,34 are linked to GJ channel closure and/or progression through the cell cycle. TRIM21 is an E3 ubiquitin ligase that forms a Skp1-Cullin-Fbox/ SCF complex regulating the ubiquitylation and turnover of p27.50−53 Cross-talk between Cx43 and E3 ligase has been shown to down-regulate levels of both Skp1 and p27.78,79 Confocal localization, IPs and SEC profiling performed in this study suggest this interaction is likely established early in the connexin life-cycle and continues during late-stage turnover from the plasma membrane. Somewhat unexpected, our results also found that Cx43 phosphorylation, not association with TRIM21 alone, regulates GJ ubiquitylation. Using a system-level approach to examine these effects enabled by SEC complex profiling, we found changes in Cx43 phosphorylation, manifested as a 83.4% increase in the slower SDS-PAGE migrating species, responsible for stimulating ubiquitin-conjugation. Monitored under both basal and EGF-stimulated conditions, our data support a role for phosphorylation inducing a strong effect on degrading GJs (Figure 7C−E). Indeed, mechanisms of substrate turnover by other RING domain E3s have been demonstrated for both sitespecific and multisite-phosphodegrons.72,74,80 For example,

while phosphorylation at individual sites stimulates a slow graded response, the synergistic phosphorylation of Sic1 at six residues is capable of inducing an ultrasensitive degradation switch.72,74 Our findings of ubiquitylated and degraded Cx43 strictly within highly phosphorylated connexin complexes at measured thresholds provides the first mechanistic evidence for a GJ phosphodegron, presumably through multisite modification. Whatever the specific mechanism and/or order of phosphate addition used, we find this pattern of phosphorylation, ubiquitylation and degradation to be reproducibly monitored by HPLC. As it will be necessary to further test these processes, isolation techniques and candidate interactions provided by this study will provide a valuable starting point to base further investigations of not only Cx43 (full list of candidate proteininteractions, Supplemental Table S1, Supporting Information) but may also be extended to other integral membrane proteins, ion channels and sites of intercellular adhesion. From this data we additionally identify at least two mechanisms of targeting ubiquitin to GJs, through direct Cx43ubiquitin conjugation and TRIM21 ubiquitylation/autoubiquitylation activity. Such identified mechanisms for targeting ubiquitin to connexin complexes, alongside alternative modes of nonlysine ubiquitylation,81 may provide alterative explanations as to why lysine-less Cx4326 appears to have normal trafficking patterns, despite the apparent decoupling of ubiquitin signals. Surprisingly, our data do not support a role for the 26S proteasome with the direct turnover of Cx43 ubiquitylates that are assembled with TRIM21. The vast majority of studies linking the proteasome to GJ degradation are based on the use of proteasomal inhibitors, such as lactacystin, MG132, ALLN, and ALLM.22,26,41,62,82−85 While these studies demonstrate functional proteasomes are needed, they do not demonstrate whether connexins, ubiquitylated or otherwise, are direct substrates for proteasome degradation.86 These findings would suggest GJs are not modified by K48-ubiquitylation. Thus the source of K48glygly peptides identified within these MS runs, alongside TRIM21 response to the 26S, would suggest proteins residing within assemblies of Cx43 may undergo selective ubiquitylation and degradation. We speculate such a mechanisms for E3 ligase activity, or possibly autoubiquitylation by TRIM21 would provide a mechanism to ensure TRIM21 is turned off after GJ phosphodegron signals are completed. Although this study strongly implicates the ubiquitin ligase activity of TRIM21 itself, at this time we cannot rule out the possibility that other E3s associated with the GJ complex may also work in collaboration with E1 UBE1 and E2 UbcH5a. In this regard, functional insights into GJ ubiquitylation should also be closely monitored in the context of the Cx43 PPxY ubiquitylation motif,87 alongside other associated E3s, including Nedd425 and Smurf-2.88 As part of research currently underway by our group, it will be important to identify of the sites of Cx43 regulation by ubiquitylation, phosphorylation and C-terminal cleavage responding to oncogenes and other mitogens that are known to impact GJ intercellular communication.

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental Figure S1: Laser scanning confocal images of TRIM21 and Cx43 labeled in mouse primary astrocyte cultures. Cx43 (green) with TRIM21 (red) together (yellow) within overlay images as indicated (white arrows). Blue: DAPI nuclear 6143



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staining. Scale bar: 10 μm. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(L.J.F.) E-mail: [email protected]; phone: (604) 822-8311; fax: (604) 822-5227. (C.C.N.) E-mail: [email protected]; phone: (604) 827-4383; fax: (604) 822-2316. Author Contributions #

L.J.F. and C.C.N. contributed equally toward senior authorship.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Heart and Stroke Foundation of BC and Yukon (C.C.N., L.J.F.), and the Canadian Stroke Network (C.C.N.), and the Canadian Institutes for Health Research (C.C.N., L.J.F.). L.J.F. and C.C.N. are the Canada Research Chairs in Quantitative Proteomics and Gap Junctions and Disease, respectively. V.C.C. was supported by a Bluma Tischler Postdoctoral Fellowship and the Heart and Stroke Foundation of Canada. A.R.K. holds an international PhD stipend from The Danish Agency for Science, Technology and Innovation. The authors would like to thank all members of the Foster and Naus labs, particularly Maxence Le Vasseur, Mike Kozoriz and Steve Bond. Mass spectrometry infrastructure used in this work was supported by the Canada Foundation for Innovation, the British Columbia Knowledge Development Fund and the BC Proteomics Network.

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