E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).
Protein ubiquitinylation plays a key role in many important cellular processes. Ubiquitinylation requires the E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and, frequently, a substrate-specific E3 ubiquitin-protein ligase. In one class of E3 ubiquitin ligases, the catalytic domain contains a zinc-binding RING finger motif. ARD1 (ADP-ribosylation factor domain protein 1), with a RING finger domain in the N-terminal region, two predicted B-Boxes, and a coiled-coil protein interaction motif immediately preceding an ADP-ribosylation factor domain at the C terminus, belongs to the TRIM (Tripartite motif) or RBCC (RING, B-Box, coiled-coil) family. The region containing the B-Boxes and the coiled-coil motif acts as a GTPase-activating protein for the ADP-ribosylation factor domain of ARD1. We report here that full-length ARD1 or the RING finger domain (residues 1-110) produced polyubiquitinylated proteins in vitro in the presence of mammalian E1, an E2 enzyme (UbcH6 or UbcH5a, -5b, or -5c), ATP, and ubiquitin. Deletion of the RING region or point mutations within the RING sequence abolished ARD1 E3 ligase activity. All data are consistent with a potential function for ARD1 as an E3 ubiquitin ligase in cells.
Vichi
Alessandro
A
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1434, USA. vichia@nhlbi.nih.gov
Payne
D Michael
DM
Pacheco-Rodriguez
Gustavo
G
Moss
Joel
J
Vaughan
Martha
M
D000818:Animals; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D025821:Polyubiquitin / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; C080681:TRIM23 protein, human; D025821:Polyubiquitin; C489963:UBE2E1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D019204:GTP-Binding Proteins