Open AccessConnexin 43 K63-polyubiquitination on lysines 264 and 303 regulates gap junction internalization
Author(s) -
Rachael M. Kells-Andrews,
Rachel Margraf,
Charles Fisher,
Matthias M. Falk
Publication year - 2018
Publication title -
journal of cell science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.384
H-Index - 278
eISSN - 1477-9137
pISSN - 0021-9533
DOI - 10.1242/jcs.204321
Subject(s) - internalization , biology , ubiquitin , connexin , phosphorylation , microbiology and biotechnology , gap junction , sumo protein , mutant , cell , biochemistry , intracellular , gene
Gap junctions (GJs) assembled from connexin (Cx) proteins allow direct cell-to-cell communication. While phosphorylation is known to regulate multiple GJ functions, much less is known about ubiquitin's role in these processes. Using ubiquitination-type specific antibodies and Cx43 lysine/arginine mutants we show that ∼8% of a GJ, localized in central plaque domains, is K63-polyubiquitinated on K264 and K303. Percentage and localization correlate well with the short turnover rate of Cxs and GJs, removal of older channels from plaque centers, and that not all Cxs in an internalizing GJ channel need to be ubiquitinated. Connexins mutated at these two sites assembled significantly larger GJs, exhibited much longer protein half-lives and were internalization impaired. Interestingly, these ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitination is triggered by phosphorylation. Phospho-specific Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282, and 255, well-known regulatory PKC and MAPK sites. Together, these novel findings suggest that the internalizing portion of channels in a GJ is K63-polyubiquitinated, ubiquitination is critical for GJ internalization, and that K63-polyubiquitination is induced by Cx phosphorylation.