Figure 6

K63 ubiquitination of E2F1 is cell cycle regulated. (a) Western blot analysis of E2F1, cyclin A and cIAP1 in U2OS cells transfected with cyclin A siRNA±cIAP1 siRNA. HSC70 was used as loading control. (b) Ubiquitination profile of E2F1 in HeLa cells co-transfected with cyclin A siRNA±cIAP1 siRNA, then 24 h later with pCMV-3HA-E2F1 and His-tagged ubiquitin wt. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes was checked by a western blot analysis. β-Actin was used as loading control. (c) Western blot analysis of the expression of 3HA-E2F1 wt or K161/164 R mutant and cyclin A in HeLa cells transfected with cyclin A siRNA. β-Actin was used as loading control. (d) Ubiquitination profile of E2F1 in HeLa cells co-transfected with cyclin A siRNA, then 24 h later with pCMV-3HA-E2F1 or the K161/164 R mutant and His-tagged K63-only ubiquitin. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes was checked by a western blot analysis. β-Actin was used as loading control. The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both samples transfected with control siRNA. (e) Western blot analysis of cIAP1 E2F1, cyclin E, Rb, phospho (S780, S807/811) Rb in U2OS cells transfected with cIAP1-encoding construct. HSC70 was used as loading control. (f) Schematic representation of the regulation of E2F1. In the G1 phase of cell cycle, E2F1 is complexed to Rb. In the S phase, E2F1 is first methylated on arginine residue par PRMT, then K63-ubiquitinated on K161/164 in a cIAP1-dependent manner, leading to stabilization and activation of the protein. In the late S, cyclin A inhibits K63 ubiquitination of E2F1, binds to and phosphorylates E2F1 and promotes UPS-mediated degradation