Figure 1

cIAP1 stabilizes and stimulates E2F1 in an E3-ubiquitin ligase activity-dependent manner. (a, b and d) E2F1 activity and expression in Hela cells (a and b) or in cIAP1^−/−/cIAP2^−/− MEFs (d) transfected with CCNE promoter-Firefly luciferase reporter plasmid, pCMV-3HA-E2F1, along with empty vector (Vector), cIAP1, cIAP1-H588A (devoid of E3-ubiquitin ligase activity) or cIAP1-F616A (lacking dimerization capacity) mutant encoding vector. HeLa cells were treated for 24 h with PYR-41 10–35 μM before analysis (a). Upper panels: E2F1 transcriptional activity was assessed in gene luciferase experiments. Luciferase activity was normalized to β-galactosidase activity and expressed as fold induction of promoter stimulated by E2F1 alone. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t-test. ***P<0.001, **0.001<P<0.01, *0.01<P<0.1, NS P>0.1. Lower panels: the expression of the constructs was analyzed by a western blot analysis. β-Actin or HSC70 are used as loading control. *Unspecific bands. (c) Ubiquitination assay performed in HeLa cells transfected with 3HA-E2F1, His-Ubiquitin encoding vectors, with an empty vector, cIAP1 or cIAP1 F616A (F/A) mutant constructs. Ubiquitinated proteins were pulled-down by using non-selective or K63-TUBEs and ubiquitinated E2F1 is revealed by using an anti-E2F1 antibody. (e) Western blot analysis of cIAP1 or 3HA-E2F1 expression in HeLa cells transfected with pCMV-3HA-E2F1 and with empty vector (Vector), cIAP1 or cIAP1-F616A mutant (dimerization defective mutant) encoding vector, treated or not with MG132 5 μM overnight. (f) In vitro ubiquitination assay of GST-E2F1 fusion protein immobilized on gluthatione sepharose and incubated with ubiquitin, E1 and E2 recombinant proteins with or without recombinant cIAP1