Molecular Mechanisms of Gap Junction Regulation by the E3 Ubiquitin Ligase WWP1

Ubiquitylation is a post-translational modification that influences a wide variety of cellular processes including protein degradation, protein subcellular localization, cell cycle progression, transcription, and DNA damage repair. Covalent attachment of the small ubiquitin molecule to a target protein involves the sequential action of three enzymes (E1, E2, and E3). In this process, substrate specificity is conferred by the E3 ligase. Our work has focused on the function of one such E3 ubiquitin ligase, WWP1. Known targets of WWP1 include cell cycle proteins, tumor suppressors, and transcription factors that promote differentiation of mesenchymal stem cells to the osteoid lineage. Recently, we have identified a novel target of WWP1 – the gap junction protein connexin (CX) 43. In particular, we found that mice overexpressing WWP1 had a 90% reduction in CX43 within the myocardium and died from ventricular arrhythmias as a consequence. CX43 is a transmembrane protein that oligomerizes to form intercellular channels which facilitate communication between adjacent cells via the transfer of small molecules. This metabolic and electrical coupling of adjacent cells plays a vital role in almost all cellular processes including growth and differentiation, cell division, and homeostasis as well as in electroconduction of the heart. Therefore, it is not surprising that CX43 is broadly expressed in nearly every cell type, and it is likely that there are commonalities underlying the regulation of CX43 in all cells that express it. Of particular interest is the fact that CX43 has a remarkably short half-life for an integral membrane protein (on the order of 1-5 hours) in all cell types examined. To investigate the molecular mechanisms involved in CX43 turnover, we used an established tissue culture system to examine the effects of changes in expression of WWP1 on the stability and subcellular localization of CX43. We found that CX43 could co-immunoprecipitate with WWP1, and this interaction was dependent on the PPXY motif of CX43. This association promoted the K27- and K29-linked polyubiquitylation of CX43 by WWP1. Co-immunoprecipitation of WWP1 with CX43 and subsequent ubiquitylation of CX43 was enhanced in the presence of phorbol 12-myristate 13-acetate (PMA) which has been reported to induce mitogen activated protein kinase (MAPK)-dependent phosphorylation and subsequent internalization of CX43 from the cell membrane to the early endosomes. WWP1-mediated ubiquitylation was found to destabilize CX43, as the overexpression of wild type WWP1 in HeLa-CX43 cells reduced the half-life of CX43 from 2 hours to less than 1 hour, while a mutant version of WWP1 lacking ubiquitin ligase activity (C866S) had no significant effect on the stability of CX43. The increased turnover of CX43 associated with the overexpression of WWP1 also significantly reduced gap junction-mediated intercellular communication. Further investigation of the role of WWP1-mediated ubiquitylation on CX43 trafficking revealed that the ligase activity of WWP1 promoted trafficking of WWP1 from the early endosome to the late endosome with subsequent delivery to the lysosome for degradation. These observations were corroborated when endogenous WWP1 was knocked down using a siRNA pool that targets WWP1. Specifically, loss of WWP1 was associated with increased levels of CX43 on the plasma membrane and with decreased trafficking of CX43 from the early endosome to the late endosome. Instead, with WWP1 knockdown, increased co-localization of CX43 with the recycling endosome marker RAB11 was noted. These data, in conjunction with our overexpression studies, suggest that WWP1 ubiquitylates CX43 in the early endosome, and this signal is required for trafficking to the lysosome for degradation. In the absence of functional WWP1, CX43 is trafficked back to the plasma membrane via an endogenous recycling pathway whose existence hitherto has been sparsely described in the literature. Collectively, this study has identified a novel role for WWP1-mediated ubiquitylation in the trafficking and lysosomal degradation of CX43 involving an atypical ubiquitin linkage. Gap junction dysregulation is associated numerous pathological conditions including arrhythmia, skin defects, cataracts and carcinogenesis. Therefore, studies like this one that elucidate the molecular mechanisms underlying the regulation of CX43 will greatly contribute towards the development of novel therapeutics