Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors

Abstract

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

FIG. 1.

Identification of E1A-interacting proteins. (A) Silver-stained image of E1A protein complexes. Protein bands corresponding to previously known and newly identified E1A-associated proteins are shown. (B) Western blot analysis of the TAP eluate. (C) Immunoprecipitation analysis of HeLa or 293 cells. The cell lysates were immunoprecipitated with anti-E1A M73 antibody (upper panel) or with anti-E1A M58 antibody (lower panel). Proteins were eluted and resolved on a 10% SDS-PAGE gel and analyzed by Western blotting.

FIG. 2.

Mapping of FOXK1/K2 and DYRK1A/HAN11 interaction domains in E1A. (A) Schematic of adenovirus exon 1 and exon 2 deletion mutants. All mutants shown in panel A express only the 243R protein. However, exon 2 deletion coordinates are based on the 289R protein. The E1A-null mutant dl312 is not depicted. (B and C) Mapping of FOXK1/K2 and DYRK1A interaction domains. HeLa cells were infected with the indicated Adv5 E1A mutants for 24 h, and the cell lysates were immunoprecipitated (IP) with anti-E1A M73 antibody, resolved on 10% SDS-PAGE, and analyzed by Western blotting with the various antibodies. (D) Mapping of the HAN11 interaction domain. HeLa cells were transfected with S-tagged HAN11. After 24 h, the cells were infected with the indicated Adv5 E1A mutants. After 24 h, the cell lysates were immunoprecipitated with anti-E1A M58 antibody and probed by Western blotting using anti-DYRK1A, anti-CtBP2, anti-S tag, anti-E1A (M58) antibodies.

FIG. 3.

Role of Ser/Thr residues of E1A on interaction with FOXK1/K2. (A) Domains of FOXK1. The FHA and DNA binding (forkhead) domains are illustrated. Numbers indicate amino acid residues that are deleted in each FOXK1 deletion mutant. (B) Interaction of FOXK1 mutants with E1A. HeLa cells were cotransfected with either vector alone or E1A (12S) and Flag-BCL-XL (a nonspecific protein control), Flag-FOXK1 wt, or mutants. The proteins were immunoprecipitated (IP) with anti-Flag antibody, and Western blots were probed with anti-E1A (M58) or anti-Flag antibodies. (C) Schematic of the E1A-FOXK1 interaction domain. Amino acids that are deleted in the E1A mutant that is deficient in FOXK1/K2 interaction (dl1132) are highlighted with brackets. E1A mutants in Ser and Thr residues are also shown. The amino acid coordinates are based on the 289R protein; however, all viruses express the 243R protein only. (D) Interaction of FOXK1/K2 with Ser/Thr mutants. HeLa cells were transfected with vectors that expressed wt E1A (12S) or indicated mutants. Cell lysates were immunoprecipitated with anti-E1A (M73 or M58) antibody and analyzed by Western blotting with the indicated antibodies. The anti-serine 219 (residue 173 in 12S) E1A antibody was used to confirm that this residue is indeed phosphorylated. (E) 293 or HeLa cells were incubated with anti-E1A antibody-coated agarose beads. Immunoprecipitates were washed and resuspended in phosphatase buffer with or without λ protein phosphatase. Samples were incubated at 30°C for 30 min. The pellets were separated by centrifugation, washed, and incubated with HeLa cell lysates from cells transfected with Flag-FOXK1 and reprecipitated. The bound protein was washed extensively, eluted, and analyzed by Western blotting with the indicated antibodies. The panel at right is a Western blot of HeLa cells either transfected with Flag-FOXK1 or untransfected (UN).

FIG. 4.

Effect of E1A C-terminal mutants on cell proliferation and Ras-cooperative transformation. (A) Effect of Adv mutants on BRK cell proliferation. The experiment was repeated in triplicates. Each data set was normalized to the percentage of cells in S phase induced by dl312. Data are the average percentage of cells in S-phase relative to the total number of cells. (B) Effect on transformation. Colonies of BRK cells transformed with plasmids expressing either E1A dl520 (which expresses 12S only) and Ras or the indicated E1A C-terminal deletion mutants and Ras. Transformed cells were stained with crystal violet and counted. The number of colonies per well was determined. The experiments were repeated at least twice, and the results of one experiment are shown in the figure. All data are represented as means ± standard deviations. *, P < 0.05. Statistical significance was examined using a Student's t test for panels A and B. (C) Effect on tumorigenesis. Athymic mice were injected subcutaneously with pooled transformed BRK cells. Transformed cells were established from cells transfected with vectors expressing Adv2 12S cDNA or mutants along with a Ras expression vector. The average tumor volume for at least four mice per group is indicated, and representative mice are pictured.

FIG. 5.

Interaction of FOXK1/K2 with HPV E6 proteins and effect of HPV21-E6 on E1A transformation. (A) FOXK1/K2 interaction domains in E1A and E6 proteins. Sequence alignments of the E1A-FOXK1/K2 interaction domain and the N-terminal domain of HPVs 21, 14, and 20 are shown. The conserved Ser/Thr residues that are critical for FOXK1/K2 interaction are underlined. (B) Interaction of FOXK1 with β-HPV E6 protein. Human 293 cells were transfected with plasmids expressing either vector or Flag-tagged E6 proteins of HPV serotypes 5, 21, 14, and 20 or HPV21 E6 mutants. Cells lysates were immunoprecipitated with anti-Flag antibody and analyzed by Western blotting. Lysates from HPV21 E6-transfected 293 cells (without immunoprecipitation) were used as markers for FOXK1/K2. (C) Inhibition of transformation by HPV21 E6. BRK cells were transfected with plasmids expressing E1A dl1132 and Ras along with HPV21 E6 or the HPV E6 S8A mutant. The number of colonies per well was determined. All data are represented as means ± standard deviations. *, P < 0.05. Statistical significance was examined using a Student's t test.

FIG. 6.

Functional domain substitution in E1A. (A) Schematic of E1A-E6. The FOXK1/K2 interaction motif of HPV21 E6 was cloned into the region that is deleted in the E1A dl1132 mutant. (B) HeLa cells were transfected with plasmids expressing vector, E1A 12S, E1A dl1132, and the E1A-E6 chimera. Cell lysates were immunoprecipitated with anti-E1A M73 antibody and analyzed by Western blotting. Lysates from HPV21 E6-transfected HeLa cells (without immunoprecipitation) were used as markers for FOXK1. (C) Transforming activity of the E1A-E6 chimeric construct. BRK cells were transformed with plasmids expressing E1A 12S and Ras, E1A dl1132 and Ras, or the E1A-E6 chimera and Ras. The number of colonies per well was determined. All data are represented as means ± standard deviations. *, P < 0.05. Statistical significance was examined using a Student's t test.

FIG. 7.

Interaction of cellular proteins with E1A. The interaction of the N-terminal region with previously known protein complexes and the functional consequences are indicated. The interaction of FOXK1/K2 and DYRK(1A/1B)/HAN11 and CtBP1/2 and the potential functional consequences are indicated.