Article Figures & Data
Figures
- Fig 1
Amphipathic peptides that interact with 16E6 confer 16E6 interaction with p53. (A) Yeast two-hybrid plasmids express the LexA DNA binding domain fused to 16E6 or 16E6 with the intervening LXXLL peptide of E6AP (406-ELTLQELLGEE-416) or the indicated mutant peptide and then a 6-amino-acid linker (GGSGGS). The 16E6 carboxy-terminal PDZ binding motif (PBM) interacts with the PDZ domain of tyrosine phosphatase PTPN3 to demonstrate expression of the LexA-E6 fusions. In the lower part of the panel are B42 transactivator fusions to E6AP containing the C843A mutation, which ablates ubiquitin ligase activity (E6AP-Ub−). The LXXLL motif, which binds 16E6, and mutant versions of the LXXLL motif are indicated. LQELS refers to the 11-amino-acid E6AP peptide sequence that has been mutated to ELTLQELSGEE, and LQEAS refers to the sequence ELTLQEASGEE. The LexA_FDELL_16E6 fusion expresses LexA fused to a 16E6-binding amphipathic peptide (MEGVFDELLGE) (23), then a 6-amino-acid linker (GGSGGS), and then 16E6. (B) LXXLL peptide interaction with 16E6 in cis blocks LXXLL peptide interaction in trans and reduces interaction with E6AP in trans. LexA reporter strains with the indicated LexA fusions were mated to strains expressing the indicated B42-transactivator fusions as previously described (5). Mated products were selected and then patched onto X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plates to assay for protein interaction, indicated by dark color development. (C) LXXLL peptide binding to 16E6 confers interaction with p53. (D) E6 interaction with non-E6AP peptide sequences can confer interaction with p53. The LexA_FDELL_16E6 fusion expresses LexA fused to a 16E6-binding amphipathic peptide (MEGVFDELLGE) (23), then a 6-amino-acid linker (GGSGGS), and then 16E6. (E) Unfused 16E6 can recruit p53 to the minimal LQELL peptide. Strains with the indicated LexA fusions to E6AP-Ub−, the indicated fragments of E6AP-Ub−, or the minimal 11-amino-acid E6 binding motif of E6AP were mated to strains expressing 16E6, p53, or the combination of 16E6 and p53. p53 does not interact with E6AP unless 16E6 is present, and the minimal 11-amino-acid LQELL peptide of E6AP is sufficient to bind E6 and recruit p53. (F) Quantitative assay of beta-galactosidase activity of yeast strains from panel E. Diploid mated yeasts from the indicated spots in part E were grown in liquid galactose medium (to induce p53 expression) for 4 h and then assayed for beta-galactosidase activity using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate as described previously (5); the asterisk indicates a P value of <0.005 by Student's t test. Results are averages from three experiments, normalized to data for spot 4B.
- Fig 2
LXXLL_16E6 associates with the p53 core DNA binding domain. (A) Diagram of p53 domains, adapted from reference 14. (B) Association of p53 domains with LXXLL-bound 16E6. LXXLL-peptide-bound 16E6 does not require the p53 transactivation domain and associates with p53-(92–393). The vertical white line indicates matched X-Gal indicator plates used in this experiment. (C) LXXLL_16E6 interacts with p53-(92-393), while the 16E6–E6AP-Ub− complex requires the full-length p53 protein. The vertical black line indicates the position of a removed irrelevant sample in the same plate, and the vertical white lines indicate additional separate but matched X-Gal indicator plates used in this experiment. (D) LXXLL peptide-bound 16E6 interaction with p53 is sensitive to missense mutations within the p53 core DNA-binding domain. The H179Q and V143A point mutants of p53 are cancer-associated mutants. p53 mutants with mutations in the transactivation domain were a gift from Jiayuh Lin (Nationwide Hospital, Columbus, OH) (11).
- Fig 3
LXXLL peptide interaction stabilizes 16E6 expression but does not target p53 for degradation in the absence of E6AP. 8B9 cells are E6AP-null mouse kidney epithelial cells transformed by adenovirus E1a and activated ras oncogenes (gift from Lawrence Banks, ICGEB, Trieste, Italy) which were cultured in Dulbecco's modified Eagle medium and 10% fetal bovine serum and transfected using polyethyleneimine at an early passage. (A) Re-expression of E6AP is required for 16E6 to efficiently target the degradation of p53. 8B9 cells were cotransfected with human p53 (0.5 μg), HA-tagged GFP as an internal transfection control (0.25 μg), 1.0 μg of untagged E6AP or E6AP_Ub−, and the indicated amount of E6 (in μg) and lysed 24 h posttransfection. Transfected human p53 was detected with human specific monoclonal antibody Ab-8 (Oncogene Science). (B) Re-expression of E6AP is required for LQELL_16E6 to efficiently target the degradation of p53. Experiments were carried out as described for panel A, but 8B9 cells were transfected with HA_LQELL_16E6 instead of WT 16E6. (C) LXXLL peptides stabilize 16E6 expression but do not target p53 degradation. 8B9 cells were transfected with the indicated plasmids as described for panel A. All the LXXLL fusions to 16E6 were epitope tagged with HA. Cotransfected LacZ was used as an internal transfection control, and HA-NS refers to a nonspecific HA-reactive cellular band used as a loading control. The white line indicates removal of an irrelevant sample. The sample was divided, with total cell lysate being used for Western blotting and the soluble fraction being immunoprecipitated with rabbit anti-HA followed by Western blotting with HA monoclonal antibody. (D and E) Epitope-tagged 16E6 can artifactually appear to target p53 degradation in an E6AP-independent manner. 8B9 cells were transfected with cDNA expression plasmids (all based on pcDNA3) using polyethyleneimine, 150 ng HA_GFP, 250 ng human p53, 650 ng E6AP or E6AP-Ub−, and the indicated amounts of wild-type 16E6 or HA-tagged 16E6 and balancing amounts of empty pcDNA3 vector to a total of 4 μg and harvested 24 h later with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. While wild-type 16E6 and LQELL_16E6 targeted only p53 degradation in the presence of wild-type E6AP, p53 was lost in lane 5 of cells transfected with HA-16E6. However, the loss of cotransfected HA_GFP in this lane suggests that the loss of p53 expression is due to cellular toxicity of overexpressed HA-16E6 in 8B9 cells. Results in panels D and E are representative of three replicate experiments.
