BC-box sequences and E4orf6 mutants. (A) Consensus BC-box sequence. (B) Putative BC-box sequences in E4orf6 and E1B55K. The sequences of two putative BC-boxes in E4orf6 and one in E1B55K have been presented with residue numbers within the proteins noted. (C) List of BC-box mutants created in E4orf6.

Effects of BC-box mutations on the activity of E4orf6. H1299 cells (A, C, and D) or H1299 cells stably expressing HA-tagged Cul5 (B) were transfected with plasmid DNAs encoding wt or mutant E4orf6. (A and B) Whole-cell lysates were immunoprecipitated (IP) with anti-E4orf6 antibody 1807 and then subjected to Western blotting analysis with anti-elongin C (ElonC) (A) or anti-HA (B) antibodies. The use of wt or mutant E4orf6 (mutants identified in Fig. 1C) or no E4orf6 (−) has been indicated at the top of each lane. The top two panels represent Western blotting analyses for elongin C or Cul5 (A and B, respectively), using shorter and longer exposures of the gel. The bottom two panels represent Western blotting analyses of whole-cell extracts for E4orf6 and elongin C levels, as indicated. (C and D) Whole-cell lysates were analyzed for p53 or Mre11 expression levels by Western blotting using anti-p53 (C) or anti-Mre11 (D) antibodies. The plasmid DNAs used to transfect cells have been indicated at the top of each figure, and Western blotting analyses have been indicated for each panel.

Analysis of complex formation using elongin C mutants. (A) Representation of elongin C deletion mutants. (B and C) Sf21 insect cells were coinfected with baculovirus vectors encoding HPC4-tagged elongin C or elongin C mutants, elongin B (ElonB), and either VHL (B) or E4orf6 (C). Sf21 cell lysates were immunoprecipitated (IP) with HPC4 monoclonal antibody, which recognizes the epitope tag on elongin C, and analyzed by Western blotting against the indicated proteins.

Requirement of E1B55K BC-box. (A) H1299 or H1299-HACul5 cells (lanes noted at bottom of the figure) were transfected with plasmid DNAs expressing E1B55K and/or E4orf6, as indicated at the top of the figure. Whole-cell lysates were either first immunoprecipitated (IP) (top two panels) using either 1807 anti-E4orf6 or anti-E1B55K antibodies as indicated on top of the figure or examined directly (bottom four panels) by Western blotting with antibodies against HA (for HA-Cul5), E1B55K, E4orf6, or elongin C (Elon C), as indicated in the figure. (B) H1299-HACul5 cells were transfected with combination of plasmid DNAs expressing wt or BC-box mutants of E1B55K (C184T) and E4orf6 (functional mutant 1, L47G), as indicated at the top of the figure. As in panel A, immunoprecipitates prepared using 1807 anti-E4orf6 antibodies or whole-cell extracts were examined by Western blotting using antibodies against HA (HA-Cul5), elongin C, E1B55K, or E4orf6, as indicated in the figure. (C) H1299 cells were transfected with the combination of plasmid DNAs expressing E4orf6 and the wt or BC-box mutant of E1B55K (C184T), as indicated at the top of the figure. Whole-cell lysates were analyzed for Mre11 expression levels by using anti-Mre11 antibodies.

Role of E1B55K in the binding and ubiquitination of p53 and the binding of Mre11. (A) p53-H1299 cells were infected with combinations of adenoviral vectors expressing p53, E1B55K, or E4orf6, as indicated at the top of the figure. Half of the cultures were treated with proteasome inhibitor (prot. inh) MG132 at 50 nM from 18 to 24 h p.i. At 24 h p.i. whole-cell extracts were immunoprecipitated (IP) using 1807 anti-E4orf6 antibodies and the precipitates were analyzed by Western blotting using anti-p53 antibodies. The presence of slower-migrating ubiquitinated forms of p53 [(Ub)n-p53] has been noted. Shorter and longer exposures (exp.) of the gel have been included, as indicated. (B) H1299 cells were transfected with plasmid DNAs expressing E4orf6 and/or E1B55K, as indicated. Whole-cell extracts were immunoprecipitated with either anti-E4orf6 or anti-E1B55K antibodies followed by Western blotting analysis using anti-Mre11 antibodies.

E4orf6-E1B55K interactions correlate with elongin C-Cul5 complex formation. (A and B) Binding of E1B55K and p53 by E4orf6 mutants. H1299 cells were transfected with plasmid DNAs expressing wt or mutant (as identified in Fig. 1C) E4orf6 and E1B55K (A) or p53 (B). Whole-cell extracts were immunoprecipitated (IP) with 1807 anti-E4orf6 antibodies, and interactions with E1B55K (A) or p53 (B) were verified by Western blotting using appropriate antibodies.

Formation of the cullin complex correlates with E4orf6 functional activity in adenovirus-infected cells. (A) Binding to elongin C (Elon C) of E4orf6 mutants in the putative zinc-binding domain. The top panel shows a summary of results obtained by Boyer and Ketner (5) with putative zinc-binding domain E4orf6 mutants. The bottom two panels show results obtained with these mutants following Western blotting analysis using anti-elongin C antibodies of immunoprecipitates (IP) prepared using 1807 anti-E4orf6 antibodies (middle panel) or 1807 anti-E4orf6 antibodies using whole-cell extracts. (B) Binding to elongin C and E1B55K of representative members (with mutant group indicated in parentheses) of E4orf6 mutants in the amphipathic α-helical region. H1299 cells were transfected with plasmid DNAs expressing wt or the indicated E4orf6 mutants and E1B55K. Whole-cell extracts were immunoprecipitated with 1807 antibodies, and interactions with E1B55K and elongin C were verified by Western blotting with appropriate antibodies.

Schematic representation of E4orf6-E1B55K-elongin-Cul5 complex formation. (A) Interaction of E4orf6 with elongin C (C) is predicted to occur via either one of the E4orf6 BC-boxes to form a complex also containing elongin B (B), Cul5 modified by addition of Nedd8, Rbx1, and an E2 ligase. (B) E1B55K interacts with substrates to be ubiquitinated and degraded, including p53, Mre11, and others, possibly those involved in mRNA export. (C) Formation of the entire complex and the positioning of the substrate for ubiquitination occur primarily through the interaction between E4orf6 and E1B55K. E4orf6 may not interact directly with Mre11 and other substrates, apart from p53. E1B55K may interact with one or more members of the elongin-Cul5 complex (denoted by ?).