NFX1-123 and poly(A) binding proteins synergistically augment activation of telomerase in human papillomavirus type 16 E6-expressing cells

Abstract

Overcoming senescence signals in somatic cells is critical to cellular immortalization and carcinogenesis. High-risk human papillomavirus (HPV) can immortalize epithelial cells in culture through degradation of the retinoblastoma protein by HPV E7 and activation of hTERT transcription, the catalytic subunit of telomerase, by the heterodimer HPV E6/E6-associated protein (E6AP). Recent work in our laboratory identified a novel repressor of hTERT transcription, NFX1-91, which is targeted for ubiquitin-mediated degradation by HPV type 16 (HPV16) E6/E6AP. In contrast, NFX1-123, a splice variant NFX1, increased expression from an hTERT promoter that was activated by HPV16 E6/E6AP. Here, we show that HPV16 E6 bound both NFX1-91 and NFX1-123 through the common central domain of NFX1 in the absence of E6AP. NFX1-123 positively regulated hTERT expression, as its knockdown decreased hTERT mRNA levels and telomerase activity and its overexpression increased telomerase activity. We identified new protein partners of NFX1-123, including several cytoplasmic poly(A) binding proteins (PABPCs) that interacted with NFX1-123 through its N-terminal PAM2 motif, a protein domain characteristic of other PABPC protein partners. Furthermore, NFX1-123 and PABPCs together had a synergistic stimulatory effect on hTERT-regulated reporter assays. The data suggest that NFX1-123 is integral to hTERT regulation in HPV16 E6-expressing epithelial cells and that the interaction between NFX1-123 and PABPCs is critical to hTERT activity.

FIG. 1.

HPV16 E6 interacted with NFX1 at its central domain. (A) Map of NFX1-91 and NFX1-123 wild-type (WT) constructs, including common and unique domains, and the truncated constructs used in GST pull-down assays. (B) FLAG-tagged NFX1-91 (N91) or FLAG-tagged NFX1-123 (N123) was coexpressed with AU1-tagged HPV16 E6 (E6) or vector control (V) in 293T cells. Twenty-four hours later, the cells were lysed and FLAG-tagged protein was immunopurified. AU1-tagged HPV16 E6 coimmunoprecipitated with both NFX1-91 and NFX1-123. (C) In vitro-translated (IVT) NFX1-91 and NFX1-123, both pulled down with GST HPV16 E6 with and without GST E6AP present. GST alone did not interact with either NFX1 isoform. (D) In vitro-translated NFX1 central domain (CD) interacted with GST HPV16 E6. (E) GST-tagged novel C termini (CT) of NFX1-91 and NFX1-123 did not interact with in vitro-translated HPV16 E6. All films were scanned in through Adobe Photoshop. IP, 5% in vitro-translated input; PD, pull down. The GST protein levels shown represent 100% input. The arrowheads indicate GST proteins.

FIG. 2.

NFX1-123 affected hTERT mRNA levels and telomerase activity. (A) TRAP assay of HFKs expressing HPV16 E6 (E6) and NFX1-123 or vector controls, LXSN and pB. Telomerase activity was seen in cells expressing HPV16 E6, and NFX1-123 increased that activity by more than threefold. Two micrograms of protein lysate was used for each sample. −, negative control, CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} lysis buffer; +, positive controls, telomerase plus cells and TSR8 synthetic template for PCR. (B) TRAP assay of HFKs expressing HPV16 E6 (E6) and shRNA against NFX1-123 (shN123) or scramble oligonucleotide as a control (scramble). Telomerase activity was reduced in cells with knockdown NFX1-123 protein. One microgram, 0.5 μg, and 0.1 μg of protein lysate were used for each sample. −, negative control, CHAPS lysis buffer; +, telomerase plus cells. (C) Western blots of cell lysates used in panel A. Expression of HPV16 E6 (E6) degraded p53 compared to vector control cells (pB) and uninfected HFKs (control). Nucleolin is shown as a loading control. (D) Western blots of cell lysates used in panel B. Expression of HPV16 E6 (E6) degraded p53 compared to uninfected HFKs (control), and shRNA against NFX1-123 (shN123) knocked down protein levels in HFKs compared to the scrambled oligonucleotide (scramble). Nucleolin is shown as a loading control. (E) Real-time PCR of NFX1-123 knockdown cells showed an 80% reduction in endogenous hTERT mRNA levels compared to scramble control cells. Error bars, 95% confidence interval. All films were scanned in through Adobe Photoshop.

FIG. 3.

NFX1-123 interacted with poly(A) binding proteins. A silver-stained gel of eluted tagged NFX1-123 in 293T cells showed multiple proteins copurifying with NFX1-123. No protein bands were seen in the vector elution alone. The gel was scanned in through Adobe Photoshop.

FIG. 4.

NFX1-123 contains a PAM2 motif, and it bound PABPCs via this domain. (A) Maps of NFX1-123, including the PAM2 motif, and the GST-tagged truncated or mutated constructs used in GST pulldowns. (*, single mutation in PAM2 motif). An alignment of the PAM2 canonical motif with NFX1 sequence is shown. Blue, amino acids most critical to PAM2-PABC interactions (36); green, common but not consensus amino acids; +, amino acids of similar charge or hydrophobicity. The phenylalanine mutated to alanine is shown in red. (B) GST pull down of truncated and mutated constructs of NFX1 showed only interaction with the N termini of NFX1 and PABPC4. A single amino acid mutation in the PAM2 motif decreased this interaction by half. 10%, 10% in vitro-translated input; PD, pulldown; N-term, GST NFX1 N terminus; N-term*, GST NFX1 N terminus PAM2 mutant; CD, GST NFX1 central domain; 91 CT, GST NFX1-91 C terminus; 123 CT, GST NFX1-123 C terminus. The GST protein levels shown represent 50% input. The arrowheads indicate GST proteins. (C) FLAG-tagged NFX1-123 (N123) or a FLAG-tagged NFX1-123 PAM2 mutant (N123*) was coexpressed with HA-tagged PABPC4 (P4) or vector control (V) in 293T cells; 24 h later, the cells were lysed and the FLAG-tagged protein was immunopurified. HA-PABPC4 coimmunoprecipitated with wild-type NFX1-123, but its interaction with the PAM2 mutant of NFX1-123 was dramatically reduced. (D) FLAG-tagged NFX1-123 (N123) or FLAG-tagged NFX1-123 with PAM2 deleted (ΔN123) was coexpressed with HA-tagged PABPC4 (P4) or vector control (V) in 293T cells. HA-PABPC4 coimmunoprecipitated with wild-type NFX1-123, and its interaction with NFX1-123 with PAM2 deleted was similar to background with mouse IgG immunoprecipitate. All films were scanned in through Adobe Photoshop.

FIG. 5.

NFX1-123 and PABPC4 synergistically augmented hTERT promoter-regulated luciferase expression through the PAM2 motif in HFKs. (A) By luciferase assay, HFKs expressing HPV16 E6 activated expression from the hTERT promoter, and NFX1-123 augmented that expression only when HPV16 E6 was present. NFX1-123 and PABPC4 coexpressed could further increase expression over NFX1-123 alone, and the PAM2 mutant of NFX1-123 decreased this effect by half. PABPC4 did not increase expression without NFX1-123 present. (B) NFX1-123 augmented activation of HPV16 E6, but NFX1-123 with PAM2 deleted (NFX1-123 ΔPAM2) did not. (C) NFX1-123 and PABPC4 did not activate hTERT promoter-driven expression without HPV16 E6. RLU, relative light units. The error bars indicate 95% confidence intervals.