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 II I human ubiquitin-conjugating enzymes (E2s), UBE2E2 and UBE2E3, the biologi cal functions of which are not known. In this study, we performed yeast two -hybrid screening for protein(s) interacting with UBE2E2, and two RING-fing er proteins, ARA54 and RNF8, were identified. Both ARA54, a ligand-dependen t androgen receptor coactivator, and RNF8 interacted with class III E2s (UB E2E2, UbcH6, and UBE2E3), but not with other E2s (UbcH5, UbcH7, UbcH10, hCd c34, and hBendless) in the yeast two-hybrid assay. The use of various delet ion 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, s howed that the UBC domain of UBE2E2 and the RING domain of these RING-finge r proteins were involved in this association. Wild-type ARA54 and RNF8, exp ressed in insect Sf9 cells, catalyzed E2-dependent autoubiquitination in vi tro, whereas the point mutated proteins showed markedly reduced activity. U biquitination of wild-type ARA54 and RNF8, expressed in COS-7 cells, was al so observed, and a proteasome inhibitor, MG132, prevented the degradation o f these wild-type proteins, but was much less effective in protecting the R ING mutants. Transfection of COS-7 cells with a green fluorescent protein c himera showed that RNF8 was localized in the nucleus, and ARA54 in both the cytoplasm and nucleus. Our results suggest that ARA54 and RNF8 possibly ac t as Ub-ligases (E3) in the ubiquitination of certain nuclear protein(s).