Figure 2

cIAP1 overexpression revealed a K63 ubiquitination of E2F1 on lysine residue 161/164. (a) Schematic representation of E2F1 protein structure indicating the lysine residues and lysine clusters. (a) Cyclin A: CDK2-binding domain; DBD: DNA-binding domain, dimer: dimerization domain; MB: marked box: Rb: Rb-binding domain. (b) Gene luciferase experiments performed in HeLa cells transfected with CCNE promoter-Firefly luciferase reporter plasmid, pCMV-HA (vector), HA-tagged-E2F1 (one single or 3HA) or HA-E2F1 mutants in which indicated K have been mutated into R along with empty vector (vector) or cIAP1-encoding vector. Luciferase activity was normalized to β-galactosidase activity and expressed as fold induction of promoter stimulated by E2F1. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t test. ***P<0.001, *0.01<P<0.1, NS P>0.1. The expression of the E2F1 constructs was checked by a western blot analysis (upper panel). β-Actin is used as loading control. (c and d) Ubiquitination assay performed in HeLa cells transfected with 3HA-E2F1 wt, K137R mutant or K161/164R mutant, His-Ubiquitin, with an empty vector or cIAP1 or cIAP1 F616A (F/A) mutant constructs. E2F1 is IP by using specific anti-E2F1 antibody and ubiquitination is revealed by using pan-ubiquitin antibody (FK2) (c), or ubiquitinated proteins were pulled-down by using K63-specific TUBEs and ubiquitinated E2F1 is revealed by using an anti-E2F1 antibody (d). (e) IP analysis of the interaction of cIAP1 with E2F1 wt or K161/164 R mutant. Hela cells were transfected with 3HA-E2F1 encoding constructs and cIAP1. cIAP1 or E2F1 were IP using anti-cIAP1 or anti-HA antibody and cIAP1-E2F1 interaction was revealed by a western blot analysis. KO: cell lysate from cIAP1^−/− KO MEFs was used to check nonspecific reactivity of the antibody