Phosphorylierung und Gap Junctions - Charakterisierung der Interaktion von Connexin 43 mit der Ubiquitin-Protein-Ligase Nedd4

The gap junction protein connexin 43 (Cx43) is a transmembrane protein with a short half-life. Degradation of Cx43 involves both the lysosome and the ubiquitin-proteasome pathway. The function of Cx43 is regulated by post-translational modifications like ubiquitination and phosphorylation. Phosphorylation of Cx43 by different kinases can affect the gap junctional intercellular communication in a positive way as well as in a negative way. E.g., phosphorylation of Cx43 at serine residues S279 and S282 is sufficient to inhibit cell to cell communication. To investigate especially the effects of the S279/S282 phosphorylation of Cx43, the polyclonal antibody SA226P was designed and prepared. SA226P is generated against a peptide containing serine residues S279 and S282 in the carboxy terminal tail of Cx43 in a phosphorylated state. Indirect immunofluorescence experiments in untreated cells revealed a faint signal in the nuclei of the cells with antibody SA226P. This observation indicates the presence of S279/S282 phosphorylated Cx43 in the nuclei. In contrast, epidermal growth factor induced S279/S282 phosphorylation was coupled to the localisation of Cx43 to gap junction plaques. Investigations to find new binding proteins of Cx43 identified the ubiquitin-protein ligase “neural precursor cell expressed, developmentally downregulated 4” (Nedd4) as a new interaction partner of Cx43. The results of the pull-down experiments with glutathione S-transferase fusion proteins of the three protein-protein interaction domains of Nedd4 (WW1, WW2 and WW3) demonstrated that WW2 and WW3 but not WW1 bind to the carboxy terminus of Cx43. The surface plasmon resonance analyses showed that the WW2 domain of Nedd4 binds specifically to the PY-motif of Cx43, but in a phosphorylation independent manner. In contrast, binding of the WW3 domain to different motifs in the carboxy terminus of Cx43 seems to be regulated by the phosphorylation state of Cx43. The in vitro interaction of Cx43 and Nedd4 was confirmed in vivo by indirect double immunofluorescence. Colocalisation of both proteins was found in multivesicular bodies. On the basis of the results with antibody SA226P it was shown that the S279/S282 phosphorylation of Cx43 plays an important role during mitosis. Colocalisation especially of S279/S282 phosphorylated forms of Cx43 with Nedd4 and ubiquitin could be detected in the centrosomes of mitotic cells. Furthermore, in mitotic cells Cx43 proteins localised at gap junction plaques were phosphorylated mainly at serine residues S279 and S282 besides other amino acid residues. Therefore, it is supposed that during mitosis phosphorylation of serine residues S279 and S282 acts as a transport signal, which results in the targeted ubiquitination of Cx43 by Nedd4 in the centrosomes. Experiments with the proteasome inhibitor lactacystin showed that exclusively phosphorylated, and mainly S279/S282 phosphorylated forms of Cx43 localised at gap junction plaques, were degraded by the proteasome. Thus, these results also indicate that a special hyperphosphorylation of Cx43 including serine residues S279 and S282 leads to internalisation of gap junctions and results in the degradation of the channels by the proteasome.