N-Terminally extended human ubiquitin-conjugating enzymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8.
We have previously cloned cDNAs encoding the N-terminally extended class III human ubiquitin-conjugating enzymes (E2s), UBE2E2 and UBE2E3, the biological functions of which are not known. In this study, we performed yeast two-hybrid screening for protein(s) interacting with UBE2E2, and two RING-finger proteins, ARA54 and RNF8, were identified. Both ARA54, a ligand-dependent androgen receptor coactivator, and RNF8 interacted with class III E2s (UBE2E2, UbcH6, and UBE2E3), but not with other E2s (UbcH5, UbcH7, UbcH10, hCdc34, and hBendless) in the yeast two-hybrid assay. The use of various deletion mutants of UBE2E2 and RING-finger proteins and two RING point mutants, ARA54 C(220)S and RNF8 C(403)S, in which the RING structure is disrupted, showed that the UBC domain of UBE2E2 and the RING domain of these RING-finger proteins were involved in this association. Wild-type ARA54 and RNF8, expressed in insect Sf9 cells, catalyzed E2-dependent autoubiquitination in vitro, whereas the point mutated proteins showed markedly reduced activity. Ubiquitination of wild-type ARA54 and RNF8, expressed in COS-7 cells, was also observed, and a proteasome inhibitor, MG132, prevented the degradation of these wild-type proteins, but was much less effective in protecting the RING mutants. Transfection of COS-7 cells with a green fluorescent protein chimera showed that RNF8 was localized in the nucleus, and ARA54 in both the cytoplasm and nucleus. Our results suggest that ARA54 and RNF8 possibly act as Ub-ligases (E3) in the ubiquitination of certain nuclear protein(s).
Ito
K
K
Department of Molecular Pathobiochemistry, Gifu University School of Medicine, Japan.
Adachi
S
S
Iwakami
R
R
Yasuda
H
H
Muto
Y
Y
Seki
N
N
Okano
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D016335:Zinc Fingers
D002352:Carrier Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D009097:Multienzyme Complexes; C118499:RNF14 protein, human; D011993:Recombinant Fusion Proteins; C106347:UBE2E2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases