Abstract
Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) was the first ubiquitin protein ligase identified to interact with connexin43 (Cx43), and its suppressed expression results in accumulation of gap junction plaques at the plasma membrane. Nedd4-mediated ubiquitination of Cx43 is required to recruit Eps15 and target Cx43 to the endocytic pathway. Although the Cx43 residues that undergo ubiquitination are still unknown, in this study we address other unresolved questions pertaining to the molecular mechanisms mediating the direct interaction between Nedd4 (WW1-3 domains) and Cx43 (carboxyl terminus (CT)). All three WW domains display a similar three antiparallel β-strand structure and interact with the same Cx43CT(283)PPXY(286)sequence. Although Tyr(286)is essential for the interaction, MAPK phosphorylation of the preceding serine residues (Ser(P)(279)and Ser(P)(282)) increases the binding affinity by 2-fold for the WW domains (WW2 > WW3 ≫ WW1). The structure of the WW2·Cx43CT(276-289)(Ser(P)(279), Ser(P)(282)) complex reveals that coordination of Ser(P)(282)with the end of β-strand 3 enables Ser(P)(279)to interact with the back face of β-strand 3 (Tyr(286)is on the front face) and loop 2, forming a horseshoe-shaped arrangement. The close sequence identity of WW2 with WW1 and WW3 residues that interact with the Cx43CT PPXY motif and Ser(P)(279)/Ser(P)(282)strongly suggests that the significantly lower binding affinity of WW1 is the result of a more rigid structure. This study presents the first structure illustrating how phosphorylation of the Cx43CT domain helps mediate the interaction with a molecular partner involved in gap junction regulation.
Highlights
Gap junction channels serve to directly interconnect the cytoplasm of neighboring cells, allowing the passage of ions, metabolites, and signaling molecules
All peaks observed in Circular Dichroism (CD) rely on the intensity and energy of transitions that depend on the and angles; the data suggest that the WW1 structure could be slightly different from the WW2 and WW3 structures
The Cx43 residues that undergo ubiquitination are still unknown, in this study, we address other unresolved questions pertaining to the molecular mechanisms mediating the direct interaction between Neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) and Cx43
Summary
Gap junction channels serve to directly interconnect the cytoplasm of neighboring cells, allowing the passage of ions, metabolites, and signaling molecules. Removal from the plasma membrane is directionally regulated as internalization of the channels within one of the coupled cells forms a double membrane macrostructure called an annular gap junction or connexosome [10]; clathrin and other endocytic adaptors have been shown to be involved in this process [11,12,13,14]. Studies using cultured cells have shown that activation of protein kinase C (PKC) or mitogenactivated protein kinases (MAPK) induces hyperphosphorylation and subsequent degradation of Cx43 (19 –21). Another type of post-translational modification universally implicated in protein degradation is ubiquitination. A proteome-wide survey by mass spectroscopy recently detected in vivo ubiquitination of Cx43 at lysine residues 9 and 303 [26], but the significance remains unclear
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