Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process

Abstract

Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.

Reorganization of Gap Junctions after Focused Ultrasound Blood–Brain Barrier Opening in the Rat Brain

“…As connexin phosphorylation of both amino acids has been correlated with increased degradation (Laird, 2005;Lampe and Lau, 2004;Leykauf et al, 2006), these results support our immunofluorescence findings and indicate that opening of the BBB with ultrasound does not result in increased connexin degradation.…”

Structural Studies of the Nedd4 WW Domains and Their Selectivity for the Connexin43 (Cx43) Carboxyl Terminus

“…Therefore, Cx43CT presents two different but overlapping WW binding motifs (PPXY and pSPXpSP) that could work cooperatively to increase the binding affinity with Nedd4 or independently to dictate which WW domain binds the Cx43CT. The possibility of Nedd4 also interacting with the class IV motif is supported by the observation that Cx43 MAPK phosphorylation after EGF stimulation increased binding to WW2 and WW3 (27). Additional unresolved questions from these studies were the location and nature of the binding motif on the Cx43CT for the WW1 and WW3 domains as well as the mechanism of differential WW domain binding caused by phosphorylation of the Cx43CT.…”

“…The neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) was the first ubiquitin E3 ligase identified to directly interact with Cx43, and its suppression by siRNA results in an accumulation of Cx43 at the membrane (12,27). Nedd4 was discovered while screening genes down-regulated during development of the central nervous system in embryonic mice but was later found to be ubiquitously expressed across cell types and species (28,29).…”

“…The WW recognition domains, named after the presence of two conserved tryptophan residues, regulate the substrate selection. Rat Nedd4 contains three WW domains, which have been shown to interact with the Cx43CT (27). Found in a wide variety of signaling proteins (30), WW domains are well folded compact polypeptides containing 38 -40 amino acids and are divided into five groups according to their binding motif preference.…”

“…Class IV binds phosphorylated (S/T)P motifs. The Cx43CT binding site for WW2 was identified and defined as a class I motif (PPXY; Cx43 residues 283-286 (27)). This finding was supported by Girão et al (12) who showed that the Cx43 mutation P283L (LPXY) reduced the binding affinity between Nedd4 and Cx43.…”