Regulation of E2F1 by APC/C Cdh1 via K11 linkage-specific ubiquitin chain formation

Abstract

E2F1 is a eukaryotic transcription factor that is known to regulate various cellular pathways such as cell cycle progression, DNA replication, DNA damage responses and induction of apoptosis. Given its versatile roles, a precise and tight regulation of E2F1 is very critical to maintain genomic stability. E2F1 is regulated both at transcriptional and posttranslational levels during cell cycle and upon DNA damage. After S phase, E2F1 is targeted for degradation and is kept at low levels or in an inactive state until the next G 1/S phase transition. Our studies show that APC/C ubiquitin ligase in conjunction with its co-activator Cdh1 (APC/C (Cdh1) ) can downregulate E2F1. We also identify an APC/C subunit APC5 that binds to E2F1 and is essential for E2F1 ubiquitination. We confirm an interaction between E2F1 and Cdh1 as well as an interaction between E2F1 and APC5 both in vivo and in vitro. In vitro GST pull-down assays have mapped the C-terminal 79 a.a. of E2F1 as Cdh1 interacting residues. Ectopically expressed Cdh1 downregulates the expression of E2F1-4. Our studies have also shown for the first time that E2F1 can be modified by K11-linkage specific ubiquitin chain formation (Ub-K11). The formation of Ub-K11 chains on E2F1 is increased in the presence of Cdh1 and accumulated in the presence of proteasome inhibitor, suggesting that APC/C (Cdh1) targets E2F1 for degradation by forming Ub-K11 chains. We also show that the effect of Cdh1 on E2F1 degradation is blocked upon DNA damage. Interestingly, Ub-K11-linked E2F1 accumulates after treatment of DNA damaging agents. The data suggest that DNA damage signaling processes do not inhibit APC/C (Cdh1) to ubiquitinate E2F1. Instead, they block the proteasomal degradation of Ub-K11-linked E2F1, and therefore lead to its accumulation.

Figure 1. E2F1 directly binds to Cdh1 via its C terminus. GST-E2F1 WT (A and B) or truncation mutants (C) were incubated with in vitro translated [S³⁵]-labeled Cdh1 or APC5 (A) or bacterially purified Cdh1 (B). E2F1-bound Cdh1 was detected by autoradiography (A) or by immunoblotting (B and C). Right panel of (C): The intensity of Cdh1 signals bound to E2F1 WT or truncation mutants as well as the amounts of GST, GST-E2F1 and GST-E2F1 truncation mutants on the corresponding GST pull-down assay were quantified using Image J software. The bound Cdh1 signals were then normalized by the abundance of each corresponding GST fusion protein in that assay. The results averaged from three independent experiments are plotted here. FL: full-length; the numbers indicate the amino acid residues of hE2F1.

Figure 2. E2F1 interacts with APC/C components Cdh1 and APC5 in vivo. HEK293 cells were transfected with plasmids expressing HA-E2F1 and APC5 (A) or GFP-Cdh1 (B), and then treated with MG132 as indicated. (A) HA-tagged E2F1 was immunoprecipitated from cell lysates with HA beads and the co-immunoprecipitated APC5 was detected by immunoblotting. (B) GFP-Cdh1 was immunoprecipitated from cell lysates using GFP antibody and the co-immunoprecipitated E2F1 was detected by immunoblotting.

Figure 3. Cdh1 downregulates E2F1 protein levels. (A) HEK293T cells were transfected with HA-E2F1 along with GFP-Cdh1 or GFP vector control. Cell lysates were prepared and subject to SDS-PAGE and immunoblot analysis as indicated. (B) To determine whether downregulation of E2F1 by Cdh1 is mediated through a post-transcriptional mechanism, HEK293T cells were transfected as in (A). Cells were harvested for mRNA isolation and E2F1 mRNA and Cdh1 mRNA were measured by real-time RT-PCR. The p value for a difference in E2F1 mRNA levels between GFP and GFP-Cdh1 samples is 0.70 (two-tailed t-test). A fraction of cells were used to prepare lysates and the lysates were analyzed by SDS-PAGE and immunoblot analysis as indicated. (C) HEK293T cells were transfected with HA-E2F1 or E2F2 or E2F3 or E2F4 along with GFP-Cdh1 or GFP control. Two days later, cells were harvested and analyzed by immunoblot analysis as indicated.

Figure 5. Cdh1-mediated degradation of E2F1 is inhibited upon DNA damage. (A) HEK293T cells were transfected with HA-E2F1 with GFP-Cdh1 (or with GFP control) and cells were either left untreated or treated with adriamycin (5 μM) for 5 h. E2F1 and GFP-Cdh1 in total lysates were analyzed by SDS-PAGE and immunoblotting. (B) HEK293T cells were transfected with HA-E2F1 with pcDNA3-Cdh1 (or with an empty vector control) and cells were untreated or treated with neocarzinostatin (NCS, 300 ng/ml). E2F1 and Cdh1 in total lysates were analyzed by SDS-PAGE and immunoblotting. The arrow indicates endogenous and overexpressed Cdh1. The lower molecular-weight band recognized by Cdh1 antibody was seen sometimes in cells transfected with untagged Cdh1 and could be a degradation product of Cdh1. (C) HEK293T cells were transfected with HA-E2F1 with GFP control (upper panels) or HA-E2F1 with GFP-Cdh1 (lower panels). The cells were then either left untreated or treated with adriamycin (5 μM) for 16 h. Next, the cells were treated with cycloheximide (20 μg/ml) for various time points. HA-tagged E2F1 and GFP-Cdh1 proteins were detected by western blot. E2F1 signals were measured by densitometry and the relative intensities compared with the respective 0 h sample are shown below each panel.

Figure 4. Cdh1 promotes K11-linkage specific ubiquitination of E2F1. (A) HEK293 cells were transfected with Myc-ubiquitin along with vectors expressing siRNA for APC5, Cdh1 or a scrambled (Scrb) sequence control. The cells were left untreated or treated with MG132. Lysates were immunoprecipitated with normal mouse IgG or a monoclonal E2F1 antibody, followed by immunoblotting to detect ubiquitinated E2F1. An aliquot of total lysates were immunoblotted as indicated. (B) HEK293 cells were transfected either with a control empty vector, E2F1 alone, or E2F1 with Cdh1. The cells were left untreated or treated with MG132. Lysates were immunoprecipitated with normal mouse IgG or a monoclonal E2F1 antibody, followed by immunoblotting with ubiquitin antibody to detect ubiquitinated E2F1. “Lysates” lane is from total cellular lysates without immunoprecipitation and serves as a positive control for ubiquitin immunoblotting. Aliquots of each input lysate were also immunoblotted as indicated (below). (C) HEK293 cells were transfected with an empty vector or HA-E2F1 along with a vector expressing siRNA for APC5, Cdh1 or a scrambled (Scrb) sequence control. Two days later, cells were harvested and lysates were immunoblotted with indicated antibodies. (D) HA-Ub-K11-specific ubiquitin was overexpressed in HEK293T cells along with GFP-Cdh1 (or GFP vector control) and Flag-E2F1 as indicated in the presence (lower panel) or absence (upper panel) of proteasome inhibitor Velcade. Lysates were processed as described in Materials and Methods, and then immunoprecipitated with anti-Flag beads. The HA-Ub-K11-conjugated E2F1 were detected by HA immunoblotting.

Figure 6. Cdh1-promoted K11-linkage-specific ubiquitination of E2F1 is enhanced and accumulated after DNA damage. HEK293T cells were transfected with Flag-E2F1, HA-Ub-K11 and GFP-Cdh1 (or GFP vector control) as indicated. The cells were either left untreated or treated with 5 μM adriamycin for 5 h before harvesting. The lysates were prepared and analyzed for E2F1 ubiquitination as described in methods section. Fractions of the total lysates were analyzed as well (lower panels).