Mechanisms of Gap Junction Phosphodegradation

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Gap junctions (GJs) are intercellular channels that allow for the exchange of low molecular weight (1kDa) ions, molecules and secondary messengers between adjacent cells. Intercellular communication permitted by GJs is critical for tissue homeostasis as it provides the most direct means for metabolic and electrical synchronization. Short half-life and demonstrated trafficking patterns within the secretory pathway consistent with complex network to maintain connexin levels, including synthesis, transport, assembly, gating and internalization from the plasma membrane. Gap junctions comprised of connexin43 (Cx43) undergo high rates of production and degradation, such that gap junctions within the human body are replaced daily (half-life 1-5.5 hr). Intercellular communication is know to regulate a diverse set of cellular and physiological processes. The structures making up gap junctions appear to be incredibly complex, with a large number of proteins supporting the assembly and turnover. Despite significant efforts, very little is known about the basic mechanisms that governing gap junction degradation. We have previously characterize the proteome of Cx43 of gap junction and provided evidence for phosphorylation-induced degradation (phosphodegron) associated with the E3 ubiquitin ligase TRIM21. The research proposed in this application will reveal the precise molecular mechanisms the turnover of these structures in responses to epidermal growth factor, a know regulator of gap junctional communication loss. The proteomic characterization of Cx43 post-translational modification, protein-protein interactions will form the core focus of this program of study.******We propose Cx43/TRIM21 is involved with gap junction internalization and loss of intercellular communication. Our plan is to: 1) identify the functional domains of TRIM21 that are responsible for interaction with Cx43; 2) conduct the in depth analysis phosphoproteomic analysis Cx43/TRIM21 complexes. The research proposed will reveal the signalling pathways and the temporal events responsible for gap junction loss. We expect that our work will reveal new concepts governing the loss of gap junction intercellular communication that will provide critical insights into how communicating gap junctions are lost in disease. At molecular resolution, the research proposed will reveal the temporal events underlying the loss of intercellular communication. Students supported by this NSERC grant will obtain expert skills in cellular proteomics, chromatography and mass spectrometry. The training of highly qualified personnel with such skills after currently in demand within multiple areas of diagnostic testing and life science research.*****