Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain.
Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.
Bruce
M Christine
MC
Program in Cell and Structure Biology, The Hospital for Sick Children, University of Toronto, 101 College Street, Toronto, ON, Canada, M5G 1L7.
Kanelis
Voula
V
Fouladkou
Fatemeh
F
Debonneville
Anne
A
Staub
Olivier
O
Rotin
Daniela
D
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D004355:Drug Stability; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000502:physiology; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D054875:Ubiquitination / Q000502:physiology*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D029867:Xenopus Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases