APC/C (Cdh1) controls the proteasome-mediated degradation of E2F3 during cell cycle exit

Abstract

E2F transcription factors regulate gene expression in concert with the retinoblastoma tumor suppressor family. These transcriptional complexes are master regulators of cell cycle progression and, in addition, control the expression of genes involved in DNA repair, G 2/M checkpoint and differentiation. E2F3 has recently attracted particular attention, because it is amplified in various human tumors. Here we show that E2F3 becomes unstable as cells exit the cell cycle. E2F3 degradation is mediated by the anaphase-promoting complex/cyclosome and its activator Cdh1 (APC/C (Cdh1) ). E2F3 interacts with Cdh1 but not Cdc20, the other APC/C activator. Enforced expression of Cdh1 results in proteasome-dependent degradation of E2F3, whereas the overexpression of Cdc20 has no effect on E2F3 turnover. Finally, silencing of Cdh1 by RNA interference stabilizes E2F3 in differentiating neuroblastoma cells. These findings indicate that the APC/C (Cdh1) ubiquitin ligase targets E2F3 for proteasome-dependent degradation during cell cycle exit and neuronal differentiation.

Figure 1. E2F3A and E2F3B are targeted for proteasome-dependent degradation in cells withdrawing from the cell cycle. Asynchronous T98G glioblastoma cells were deprived of serum for the indicated times. Where indicated, the proteasome inhibitor MG132 (10 μM) was added for the last 5 h. Cells were collected and lysed. Whole cell extracts were analyzed by immunoblotting with antibodies for the indicated proteins. Skp1 was blotted as a loading control.

Figure 2. Degradation of E2F3A and E2F3B is stimulated by Cdh1, but not by Cdc20. T98G cells were co-transfected with plasmids expressing HA-tagged E2F3A, HA-tagged E2F3B or HA-tagged Skp2, along with FLAG-tagged Cdc20, FLAG-tagged Cdh1 or an empty vector (EV). A plasmid expressing EGFP was co-transfected as a marker of transfection. Cells were lysed and E2F3A (A), E2F3B (B), Skp2 (C) together with Cdh1 or Cdc20 were detected by immunoblotting using anti-HA and anti-FLAG antibodies, respectively. Actin was blotted as a loading control. The asterisks (*) indicate nonspecific bands. Graphs on the right show quantification of E2F3A (A), E2F3B (B) and Skp2 (C) expression. The value given for the amount of protein present in the control sample (EV) was set as 1 (n = 3).

Figure 3. Cdh1-dependent degradation of E2F3 is prevented by proteasomal inhibition. T98G cells were co-transfected with plasmids expressing either HA-tagged E2F3A or HA-tagged E2F3B, along with either FLAG-tagged Cdh1 or an empty vector (EV). A plasmid expressing EGFP was co-transfected as a marker of transfection. Where indicated, cells were treated with 10 μM MG132 for 5 h before harvesting. Cells were lysed and E2F3A (A) and E2F3B (B), together with Cdh1, were detected by immunoblotting using anti-HA or anti-FLAG antibodies, respectively. Actin was blotted as a loading control. Graphs on the right show quantification of E2F3A (A), E2F3B (B) expression. The value given for the amount of protein present in the control sample (EV) was set as 1 (n = 3).

Figure 4. Cdh1, but not Cdc20, interacts with E2F3A and E2F3B. (A) HEK293T cells were transfected with either empty vector (EV), FLAG-tagged Cdc20 or FLAG-tagged Cdh1. The proteasome inhibitor MG132 (10 μM) was added 5 hours before harvesting. Cells were lysed and whole cell extracts (WCE) were subjected either to immunoblotting or immunoprecipitation (IP) with anti-FLAG resin and subsequent immunoblotting for the indicated proteins. (B) Alignment of amino acid regions of putative destruction box motifs in E2F3A with the D-box motifs of known substrates of APC/C^Cdh1. (C) HEK293T cells were transfected with FLAG-tagged Cdh1 along with the indicated Myc-tagged E2F3A and E2F3B mutants. Cells were lysed and whole cell extracts were subjected either to immunoblotting or immunoprecipitation (IP) with anti-Myc resin and subsequent immunoblotting for the indicated proteins.

Figure 5. E2F3 is ubiquitylated by APC/C^Cdh1. In vitro ubiquitylation assay of ³⁵S-labeled, in vitro translated E2F3A was performed with immunopurified APC/C, E1, UBCH10 and ubiquitin in the presence or absence of Cdh1 translated in vitro. Samples were incubated at 30°C for the indicated times and analyzed by SDS-PAGE followed by autoradiography. The bracket on the right side marks a ladder of bands corresponding to polyubiquitylated E2F3A.

Figure 6. Cdh1 silencing stabilizes E2F3 during neuronal differentiation. (A) SK-N-SH neuroblastoma cells were treated with 10µM retinoic acid (RA) to induce neuronal differentiation. At the indicated times, cells were collected and lysed. Whole cell extracts were analyzed by immunoblotting with antibodies for the indicated proteins. Phase contrast images of all samples were taken at 10X magnification before harvesting (B). (C) Cdh1 silencing stabilizes E2F3A and E2F3B during neuronal differentiation. SK-N-SH cells were transfected twice with siRNA targeting a non-relevant mRNA (control siRNA), Cdh1 mRNA or Cdc20 mRNA. After transfection, 10 µM retinoic acid was added to induce neuronal differentiation. Cells were collected at the indicated time points and analyzed by immunoblotting with antibodies for the indicated proteins. Actin was blotted as a loading control.