Both BC-Box Motifs of Adenovirus Protein E4orf6 Are Required To Efficiently Assemble an E3 Ligase Complex That Degrades p53

Abstract

Small DNA tumor viruses typically encode proteins that either inactivate or degrade p53. Human adenoviruses encode products, including E4orf6 and E1B55K, that do both. Each independently binds to p53 and inhibits its ability to activate gene expression; however, in combination they induce p53 degradation by the ubiquitin pathway. We have shown previously that p53 degradation relies on interactions of E4orf6 with the cellular proteins Cul5, Rbx1, and elongins B and C to form an E3 ligase similar to the SCF and VBC complexes. Here we show that, like other elongin BC-interacting proteins, including elongin A, von Hippel-Lindau protein, and Muf1, the interaction of E4orf6 is mediated by the BC-box motif; however, E4orf6 uniquely utilizes two BC-box motifs for degradation of p53 and another target, Mre11. In addition, our data suggest that the interaction of E1B55K with E4orf6 depends on the ability of E4orf6 to form the E3 ligase complex and that such complex formation may be required for all E4orf6-E1B55K functions.

Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription

“…In agreement with the results described before, the E1B-NES mutant protein degraded endogenous Mre11 comparable to wt 55K (Figure 6c, compare lane 6 with 4), whereas transfection of pE1B-C454S/C456S reproducibly had no or only minor effects on Mre11 steadystate concentrations (Figure 6c, lane 5). Significantly, the same amino-acid changes did not interfere with the complete degradation of ectopically expressed p53 (Figure 6c, lanes 5 and 6), excluding the possibility that these mutations eliminate the E4orf6-dependent assembly of a high-molecular-weight E3 ubiquitin ligase complex necessary for the polyubiquitination of p53 and presumably Mre11 (Blanchette et al, 2004). Taken together, these data strongly suggest that amino acids C454 and C456 participate in the binding to Mre11 and demonstrate that active nuclear export of E1B-55K by its NES is not required for efficient degradation of p53 and Mre11 in human H1299 cells.…”

“…Several conserved cysteine and histidine residues in Ad5 E1B-55K contribute to efficient transformation of primary rat epithelial cells in cooperation with Ad5 E1A To identify further regions in the Ad5 E1B-55K oncoprotein required for transformation of primary mammalian cells, we generated a panel of mutants containing single-or double-amino-acid substitutions in different segments of the 55K polypeptide, including a predicted elongin B-and C-binding domain (Blanchette et al, 2004), a proposed zinc-finger motif (Flint and Gonzalez, 2003) and several other clustered histidine and cysteine residues, some of which are conserved in E1B-55K proteins from human subgroup A to F adenoviruses (Figure 1). These were tested for their ability to (i) inhibit p53-stimulated transcription in plasmid-transfected H1299 cells, (ii) transform primary baby rat kidney (BRK) cells in cooperation with Ad E1A, (iii) produce stable 55K proteins in transformed BRK lines and (iv) form a single E1B/p53-positive cytoplasmic perinuclear body, a characteristic of all subgroup C Ad2/5-transformed mammalian cells ( Figure 2).…”

“…Consistent with the results described before (Table 2 in Supplementary data) E1B mutants C184T and C454S/C456S blocked p53 induction of the Cyclin G promoter nearly as efficiently as the wt protein, whereas co-transfection of pE1B-L180A/C184S resulted in an only 20-40% reduction of luciferase activity (Figure 3a). The observed increase in promoter activity is likely due to the reduced expression levels of the E1B-L180A/C184S mutant ( Figure 3b, lane 3), indicating that amino-acid changes in the elongin B-and C-binding domain motif (C184T and L180A/C184S) differentially affect the stability of the Ad protein in transiently transfected and stably transformed cells ( Figure 3a; Blanchette et al, 2004). Co-transfection of plasmids encoding wt E1B-55K and the mutants fused to the Gal4 DNA-binding domain efficiently repressed transcription of the reporter plasmid pGal4TK-Luc (Figure 3c).…”

Viral-mediated stabilization of AU-rich element containing mRNA contributes to cell transformation

“…This a-helix structure is known to be essential for the oncogenic activities of E4orf6, including cooperative focus formation with E1A and promotion of tumor growth in nude mice (Nevels et al, 2000). In addition, the same structure is also required for E4orf6 to assemble into the E3 ubiquitin ligase complex with E1B55k (Blanchette et al, 2004). To analyze this region further, we produced additional E4orf6 mutants, E4orf6 L245P and E4orf6 R248E (Figure 2a), which possess amino-acid substitutions in their a-helices, and then examined their in vivo interactions with pp32.…”

“…As the a-helix structure is also thought to be important for E3 ubiquitin ligase activity mediated by E4orf6 (Orlando and Ornelles, 2002;Blanchette et al, 2004), we examined whether another E4orf6 mutant that has lost the ability to assemble into the E3 ligase complex is able to stabilize ARE-mRNA. To confirm this, we constructed the E4orf6 (BCÀ) mutant by introducing specific point mutations in BC boxes 1 and 2 (Figure 2a), which are necessary for E4orf6 to associate with Cullin5, a complex containing E3 ubiquitin ligase bound with E1B55k, using mutant adenovirus H5pm4139 (a generous gift from T Dobner) as a template (Blanchette et al, 2008).…”