Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: 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 μ M 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 μ M 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

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Ubiquitin Proteomics, Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Western Blot, Control, In Vitro, Incubation, Recombinant

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: 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

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Over Expression, Ubiquitin Proteomics, Residue, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Expressing, Construct, Western Blot, Control, Mutagenesis

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 is required for DNA damage-induced stabilization of E2F1. ( a ) Western blot analysis of E2F1 and cIAP1 in U2OS cells transfected with control or cIAP1-siRNA and treated with indicated concentration of etoposide for 6 h. β -Actin was used as loading control. ( b ) Ubiquitination profile of E2F1 in HeLa cells transfected with cIAP1 siRNA or cIAP1-encoding construct and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10 μ M etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes and the efficiency of siRNA were checked by a western blot analysis (input). β -Actin was used as loading control. ( c ) Ubiquitination assay performed in HeLa cells transfected with control or cIAP1 siRNA and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10 μ M etoposide. Ubiquitinated proteins were pulled-down by using K63 specific TUBEs and ubiquitinated E2F1 is revealed by using E2F1 antibody. ( d ) Western blot analysis of E2F1 in U2OS cells transfected with 3HA-E2F1 or 3HA-E2F1-K161/164R mutant and treated with indicated concentration of etoposide for 6 h. β -Actin was used as loading control. ( e ) Ubiquitination of E2F1 in HeLa cells transfected with pCMV-3HA-E2F1 or pCMV-3HA-E2F1 K161/164R, and with His-tagged ubiquitin wt, and then treated for 6 h with 10 μ M etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both untreated samples. ( f ) U2OS cells were transfected with cIAP1 siRNA or treated with 17 nM GDC-0152 for 1 h, and then treated with 10 μ M etoposide for 48 h. tp73 mRNA expression was measured by RT-qPCR. UT: untreated cells. Results were normalized to hprt mRNA and expressed relatively to control untreated cells. Mean±S.D. of three independent experiments

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Western Blot, Transfection, Control, Concentration Assay, Ubiquitin Proteomics, Construct, Expressing, Mutagenesis, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: PRMT-mediated arginine methylation of E2F1 is required for regulation of E2F1 by cIAP1. ( a ) Western blot analysis of E2F1 and cIAP1 in HeLa cells transfected with 3HA-E2F1 and cIAP1-encoding constructs, and treated for 24 h with 100 μ M PRMT inhibitor AMI-1. HSC70 was used as loading control. ( b ) Ubiquitination of E2F1 in HeLa transfected with pCMV-3HA-E2F1, pCI-cIAP1, and with His-tagged ubiquitin wt, and then treated for 16 h with 50 μ M AMI-1±10 μ M etoposide for 6 h. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using pan anti-ubiquitin antibody (FK2). The expression of the transgene is checked by western blot. β -Actin was used as loading control. ( c ) E2F1 transcriptional activity as measured by using a gene reporter luciferase assay performed in Hela cells transfected as in a in the presence of CCNE promoter-Firefly luciferase reporter plasmid and treated with increasing concentration of AMI-1 for 24 h. Results are expressed as average±S.D. from at least three experiments. Statistical analysis performed using Student’s t- test. *** P <0.001, **0.001< P <0.01, NS P >0.1. ( d ) Ubiquitination profile of E2F1 in HeLa transfected with Control (Co), PRMT-1 or PRMT-5-siRNA and pCMV-3HA-E2F1, pCI-cIAP1, and with His-tagged ubiquitin wt. E2F1 was IP using anti-HA antibody and ubiquitin revealed using pan anti-ubiquitin antibody (FK2). The expression of the transgene and the efficiency of siRNA were checked by western blot analysis. β -Actin was used as loading control

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Methylation, Western Blot, Transfection, Construct, Control, Ubiquitin Proteomics, Expressing, Activity Assay, Luciferase, Plasmid Preparation, Concentration Assay

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: 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

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Control, Expressing, Mutagenesis, Construct, Methylation, Residue, Activation Assay