ABSTRACT
Gap junctions (GJs) assembled from connexin (Cx) proteins play a pivotal role in cell-to-cell communication by forming channels that connect the cytosols of adjacent cells. Connexin 43, the best-studied Cx, is ubiquitously expressed in vertebrates. While phosphorylation is known to regulate multiple aspects of GJ function, much less is known about the role ubiquitination plays in these processes. Here we show by using ubiquitination-type specific antibodies and Cx43 lysine (K) to arginine (R) mutants that a portion of Cx43 in GJs can become K63-polyubiquitinated on K264 and K303. Relevant Cx43 K/R mutants assembled significantly larger GJ plaques, exhibited much longer protein half-lives and were internalization impaired. Interestingly, ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitination may be triggered by phosphorylation. Phospho-specific Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282, and 255, well-known regulatory PKC and MAPK phosphorylation sites. Together, these novel findings suggest that upon internalization, some Cx43 in GJs becomes K63-polyubiquitinated, ubiquitination is critical for GJ internalization, and that K63-polyubiquitination may be induced by Cx phosphorylation.
Summary Statement Here we show that connexin 43 in gap junctions becomes K63-poly ubiquitinated on lysines 264 and 303 and its requirement for gap junction endocytosis. These novel findings significantly contribute to our understanding of GJ turnover and patho-/physiology.
- AGJ
- annular gap junction
- AMSH
- associated molecule with the SH3 domain of STAM
- CME
- clathrin-mediated endocytosis
- Cx
- Connexin
- Cx43
- Connexin 43
- DUB
- deubiquitinase
- GJ
- gap junction
- MonoUb
- monoubiquitin
- Nedd4-1
- neural precursor cell expressed developmentally down-regulated protein 4-1
- PM
- plasma membrane
- PolyUb
- polyubiquitin
- TPA
- 12-O-Tetradecanoylphorbol 13-Acetate
- TX-100
- Triton X-100
- RT
- room temperature
- Ub
- ubiquitin