Calcium activates Nedd4 E3 ubiquitin ligases by releasing the C2 domain-mediated auto-inhibition.
Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-beta. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination.
Wang
Jian
J
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, PA, USA.
Peng
Qisheng
Q
Lin
Qiong
Q
Childress
Chandra
C
Carey
David
D
Yang
Wannian
W
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D053503:Epithelial Sodium Channels / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D016296:Mutagenesis; D000075702:Nedd4 Ubiquitin Protein Ligases; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D011993:Recombinant Fusion Proteins; C507048:SCNN1B protein, human; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D002118:Calcium