Binding of Human Papillomavirus Type 16 E6 to E6AP Is Not Required for Activation of hTERT

). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule. P apillomavirus E6 oncoproteins interact with target cellular proteins through docking on short peptides often containing the sequence LXXLL (4, 6, 26). The cancer-associated human papillomavirus type 16 (HPV-16) protein E6 (16E6) binds to an LXXLL motif (LQELL) on the cellular E3 ubiquitin ligase E6AP (10); 16E6-E6AP recruits and ubiquitinates p53. Neither 16E6 nor E6AP interacts alone with p53 (8-10, 17, 18) (20). A form of E6AP with a C833A mutation still binds E6 and p53 but fails to ubiquitinate or degrade p53 (19).The necessity of E6AP for p53 degradation by 16E6 is controversial. While E6AP is required for in vitro degradation systems (9) and immortalized mouse fibroblasts (5), E6AP-null mice that express 16E6 in the skin do not accumulate p53 when irradiated, and mouse embryo kidney cells from those E6AP-null mice are reported to lose p53 upon overexpression of 16E6 (12,22). Further, ubiquitin-independent degradation of p53 by E6 has been described (2). These disparate observations raise the questions of whether of E6AP is necessary for degradation and how the specificity of 16E6 for p53 is determined.16E6 binding to the LQELL peptide alone recruits p53 to 16E6 in the absence of full-length E6AP. Our previous studies in yeast (Saccharomyces cerevisiae) had shown that p53 was very weakly recruited by LexA fusions to 16E6, but coexpression of LexA_16E6 with the E6AP C833A mutant (here termed E6AP-Ub Ϫ , for ubiquitin ligase negative) strongly recruited p53 to 16E6 (5). We reasoned that the binding of 16E6 solely to the minimal LQELL peptide of E6AP or similar LXXLL peptides might be sufficient to induce a p53-binding conformation of 16E6. To test this, we fused the 10-residue E6AP LQELL peptide between LexA and 16E6 to provide 16E6 with an LQELL docking site in cis; we similarly fused a mutated LXXLL peptide (LQEAS) between LexA and 16E6 (Fig. 1A). We tested interactions of these LexA fusions by yeast two-hybrid assays with a B42 transactivator-tagged LQELL peptide, B42_E6AP-Ub Ϫ , or with B42_E6AP-Ub Ϫ molecules where LQELL was progressively mutated from LQELL to LQELS or LQEAS. Fusion of the LQELL peptide in cis to 16E6 blocked interaction with B42_LQELL peptide in trans as expected, and mutation of LexA_LQELL_16E6 to LexA_LQEAS_16E6 restored interactions in trans (Fig. 1B, spots 4C and 4D). This is consistent with the LQELL peptide binding in cis to the 16E6 portion of the fusion protein and blocking trans interaction with the B42-fused peptide. Interaction of LQELL_16E6 with B42_E6AP-Ub Ϫ in trans was not blocked, indicating that the interaction of 16E6 with the full E6AP protein is more robust than the interaction with the free LQELL peptide but is still dependent upon an in...

supporting

confidence: 71%

Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

Ansari

Brimer

Pol

2012

“…Myc is known to be a direct activator of telomerase in both human keratinocytes and fibroblasts (4,5,28,61), and our previous studies have shown that E6 and Myc physically interact and bind to the hTERT promoter (30,51,58). Indeed, the presence of Myc is required for E6-mediated induction of the hTERT promoter.…”

Section: E6mentioning

confidence: 88%

Cell-Restricted Immortalization by Human Papillomavirus Correlates with Telomerase Activation and Engagement of the hTERT Promoter by Myc

Dakić

Chen

et al. 2008

The high-risk human papillomaviruses (HPVs) are the causative agents of nearly all cervical cancers and are etiologically linked to additional human cancers, including those of anal, oral, and laryngeal origin. The main transforming genes of the high-risk HPVs are E6 and E7. E6, in addition to its role in p53 degradation, induces hTERT mRNA transcription in genital keratinocytes via interactions with Myc protein, thereby increasing cellular telomerase activity. While the HPV type 16 E6 and E7 genes efficiently immortalize human keratinocytes, they appear to only prolong the life span of human fibroblasts. To examine the molecular basis for this cell-type dependency, we examined the correlation between the ability of E6 to transactivate endogenous and exogenous hTERT promoters and to immortalize genital keratinocytes and fibroblasts. Confirming earlier studies, the E6 and E7 genes were incapable of immortalizing human fibroblasts but did delay senescence. Despite the lack of immortalization, E6 was functional in the fibroblasts, mediating p53 degradation and strongly transactivating an exogenous hTERT promoter. However, E6 failed to transactivate the endogenous hTERT promoter. Coordinately with this failure, we observed that Myc protein was not associated with the endogenous hTERT promoter, most likely due to the extremely low level of Myc expression in these cells and/or to differences in chromatin structure, in contrast with hTERT promoters that we found to be activated by E6 (i.e., the endogenous hTERT promoter in primary keratinoctyes and the exogenous hTERT core promoter in fibroblasts), where Myc is associated with the promoter in either a quiescent or an E6-induced state. These findings are consistent with those of our previous studies on mutagenesis and the knockdown of small interfering RNA, which demonstrated a requirement for Myc in the induction of the hTERT promoter by E6 and suggested that occupancy of the promoter by Myc determines the responsiveness of E6 and the downstream induction of telomerase and cell immortalization.The high-risk papillomaviruses (HPVs; e.g., HPV type 16 and HPV-18) are associated with anogenital carcinomas (65, 66), laryngeal carcinomas, and head and neck carcinomas (15). These HPVs carry two oncogenes, E6 and E7, which are retained and expressed in HPV-positive cervical cancers (1, 2, 50) and are required for maintenance of the tumorigenic phenotype (35, 36). The E6 and E7 proteins were first identified as targeting the p53 and Rb tumor suppressor pathways in host cells (8,9,35,36,47,48), thereby disrupting cell cycle controls.Telomerase is a specialized reverse transcriptase that synthesizes repeat DNA sequences at the ends of chromosomes termed telomeres (17). The absence of telomerase activity in most normal human cells results in the progressive shortening of telomeres with each cell division (19,56,60), eventuating in growth arrest or replicative senescence (6,19). In contrast to most human somatic cells, immortalized and cancer cells contain detectable telomerase ac...

“…Estrogen, STAT3, and c-Myc have all been shown to activate hTERT (32,34,63). HPV type 16 (HPV16) E6, along with E6-associated protein (E6AP), an E3 ubiquitin ligase, activates transcription of hTERT in epithelial cells (16,23,39), although there has been evidence of E6AP-independent effects by E6 (51). Activation of hTERT by E6-E6AP requires two E boxes found at the hTERT promoter flanking the transcriptional start site (55,58,60).…”

mentioning

confidence: 99%

NFX1-123 Increases hTERT Expression and Telomerase Activity Posttranscriptionally in Human Papillomavirus Type 16 E6 Keratinocytes

Katzenellenbogen

Vliet-Gregg²,

et al. 2009

High-risk human papillomavirus (HPV) E6 protein induces telomerase activity through transcriptional activation of hTERT, the catalytic subunit of telomerase. HPV type 16 (HPV16) E6 interacts with two splice variants of NFX1 to increase hTERT expression. NFX1-91 is a transcriptional repressor of hTERT that is polyubiquitinated and targeted for degradation by HPV16 E6 in concert with E6-associated protein. We previously showed that NFX1-123 augments hTERT expression through binding to cytoplasmic poly(A) binding proteins (PABPCs). In this study, we determined that unlike NFX1-91, NFX1-123 is a cytoplasmic protein that colocalized with PABPCs but does not shuttle with PABPCs between the nucleus and cytoplasm. NFX1-123 requires both its PAM2 motif, with which it binds PABPCs, and its R3H domain, which has putative nucleic acid binding capabilities, to increase hTERT mRNA levels and telomerase activity in keratinocytes expressing HPV16 E6. In keratinocytes expressing HPV16 E6 and overexpressing NFX1-123, there was increased protein expression from in vitro-transcribed RNA fused with the 5 untranslated region (5 UTR) of hTERT. This posttranscriptional increase in expression required the PAM2 motif and R3H domain of NFX1-123 as well as the coexpression of HPV16 E6. NFX1-123 bound endogenous hTERT mRNA and increased its stability in HPV16 E6-expressing human foreskin keratinocytes, and NFX1-123 increased the stability of in vitro-transcribed RNA fused with the 5 UTR of hTERT. Together, these studies describe the first evidence of posttranscriptional regulation of hTERT, through the direct interaction of the cytoplasmic protein NFX1-123 with hTERT mRNA, in HPV16 E6-expressing keratinocytes.Human papillomavirus (HPV) is a double-stranded DNA tumor virus, and persistent high-risk HPV (HR HPV) infections are associated with cervical and other anogenital cancers (3, 8-10, 19, 41). Dividing epithelial cells are needed for HPV DNA replication and amplification, but differentiated cells are needed for full HPV genome expression. Therefore, HPV drives the upper layers of stratified squamous epithelium to continue to divide as they differentiate, and the HR E6 and E7 proteins are critical to the programmatic disruption of normal epithelium, cellular immortalization in culture, and cervical cancer in mouse models (48, 52). The HR HPV E7 protein drives cells to continue through S phase by targeting pocket proteins for degradation (22, 66), thus allowing the E2F transcriptional factor to activate S-phase genes for DNA replication. The HR HPV E6 protein has several targets in epithelial cells. HR HPV E6 targets p53 for degradation (50), blocking apoptotic and cellular senescence signals due to DNA damage. HR HPV E6 also affects PDZ domain-containing proteins, such as MUPP-1, Scribble, MAGI-1, -2, and -3, PTPN3, and hDlg (17,24,30,36,44,56). Finally, HR HPV E6 blocks cellular senescence signals by activating telomerase (31, 58). The degradation of retinoblastoma protein by E7 and the activation of telomerase by E6 are two critical ste...