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Regulation of TopBP1 oligomerization by Akt/PKB for cell survival

2006; Springer Nature; Volume: 25; Issue: 20 Linguagem: Inglês

10.1038/sj.emboj.7601355

1460-2075

Kang Liu, Jason C. Paik, Bing Wang, Fang‐Tsyr Lin, Weei-Chin Lin,

Ubiquitin and proteasome pathways

Article28 September 2006free access Regulation of TopBP1 oligomerization by Akt/PKB for cell survival Kang Liu Kang Liu Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Jason C Paik Jason C Paik Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Bing Wang Bing Wang Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Fang-Tsyr Lin Fang-Tsyr Lin Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Weei-Chin Lin Corresponding Author Weei-Chin Lin Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Kang Liu Kang Liu Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Jason C Paik Jason C Paik Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Bing Wang Bing Wang Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Fang-Tsyr Lin Fang-Tsyr Lin Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Weei-Chin Lin Corresponding Author Weei-Chin Lin Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA Search for more papers by this author Author Information Kang Liu1, Jason C Paik2, Bing Wang1, Fang-Tsyr Lin2 and Weei-Chin Lin 1,2 1Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 2Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA *Corresponding author. Division of Hematology and Oncology, Department of Medicine, 520A Wallace Tumor Institute, University of Alabama, 1530 3rd ave S, Birmingham, AL 35294-3300, USA. Tel.: +1 205 934 3979; Fax: +1 205 975 6911; E-mail: [email protected] The EMBO Journal (2006)25:4795-4807https://doi.org/10.1038/sj.emboj.7601355 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Regulation of E2F1-mediated apoptosis is essential for proper cellular growth. This control requires TopBP1, a BRCT (BRCA1 carboxyl-terminal) domain-containing protein, which interacts with E2F1 but not other E2Fs and represses its proapoptotic activity. We now show that the regulation of E2F1 by TopBP1 involves the phosphoinositide 3-kinase (PI3K)–Akt signaling pathway, and is independent of pocket proteins. Akt phosphorylates TopBP1 in vitro and in vivo. Phosphorylation by Akt induces oligomerization of TopBP1 through its seventh and eighth BRCT domains. The Akt-dependent oligomerization is crucial for TopBP1 to interact with and repress E2F1. Akt phosphorylation is also required for interaction between TopBP1 and Miz1 or HPV16 E2, and repression of Miz1 transcriptional activity, suggesting a general role for TopBP1 oligomerization in the control of transcription factors. Together, this study defines a novel pathway involving PI3K–Akt–TopBP1 for specific control of E2F1 apoptosis, in parallel with cyclin–Cdk–Rb for general control of E2F activities. Introduction The balance between proliferation and cell death is delicately controlled throughout growth and development. Central to control of proliferation is the Rb/E2F pathway. The Rb tumor suppressor protein controls cellular proliferation through its ability to bind and repress E2F activity. E2F is a family of transcription factors, including eight members, E2F1–E2F8 (Attwooll et al, 2004; Dimova and Dyson, 2005). E2Fs activate a large array of genes that encode proteins important for DNA replication and cell cycle progression. Although E2Fs are important for cell growth, de-regulated E2F1 is able to induce apoptosis. Among the E2F family members, E2F1 has the unique ability to trigger apoptosis when expressed in the absence of growth factors (Hallstrom and Nevins, 2003). Although E2F2 and E2F3 also induce apoptosis to a lesser extent than E2F1 in some experiments, their proapoptotic activity is mediated through transactivation of E2F1 (Lazzerini Denchi and Helin, 2005). Therefore, E2F1 possesses the unique ability to promote both proliferation and death, and these activities must be under tight control for proper cellular proliferation. As the level of E2F1 surges during G1/S transition and S phase of each cell cycle, the proapoptotic activity of E2F1 must be blocked to allow cell cycle progression. The control of E2F1 apoptotic activity requires the action of the Ras–phosphoinositide 3-kinase (PI3K)–Akt pathway, because constitutively active Akt/protein kinase B (PKB) (referred hereafter as Akt) attenuates E2F1-induced apoptosis and a PI3K inhibitor abrogates serum-mediated suppression of E2F1 apoptosis (Hallstrom and Nevins, 2003). Nevertheless, the mechanism of this Akt-mediated control of E2F1 remains unclear. Recently, we have identified an E2F1-interacting protein, TopBP1 (topoisomerase II β-binding protein), that inhibits E2F1-dependent apoptosis during normal growth and DNA damage (Liu et al, 2003, 2004). Through its sixth BRCT domain, TopBP1 interacts with and represses E2F1 but not other E2F factors. The repression is mediated through recruitment of the Brg1/Brm chromatin-remodeling complex by TopBP1 to E2F1-responsive promoters, thereby repressing the transcriptional activity of E2F1. The interaction occurs not only after DNA damage but also during G1/S transition, and this regulation is crucial for the control of E2F1-dependent apoptosis during normal proliferation and DNA damage. The selectivity of TopBP1 toward E2F1, but not E2F2 or E2F3, would allow E2F2 and E2F3 to function and induce S-phase entry while E2F1 apoptosis is inhibited. Therefore, TopBP1 appears to be the key regulator for E2F1. Whether the PI3K–Akt signaling pathway controls E2F1 through TopBP1 is the subject of investigation in this study. TopBP1 is an evolutionally conserved BRCT domain-rich protein that appears to be involved in DNA replication, DNA damage checkpoint response and transcriptional regulation (Garcia et al, 2005). Human TopBP1 contains eight BRCT domains, whereas its yeast homologs, Cut5/Rad4 (Schizosaccharomyces pombe) and Dpb11 (Saccharomyces cerevisiae), contain only four. The homology between TopBP1 and Cut5/Rad4 or Dpb11 lies at its BRCT domains 1–2, and 4–5. The interaction between Rad9 and the BRCT domains 4–5 of TopBP1 is believed to recruit TopBP1 to DNA damage sites and activate the Chk1 checkpoint response (Greer et al, 2003; Furuya et al, 2004). The additional carboxyl BRCT domains of metazoan TopBP1 are responsible for interactions with transcription factors, such as E2F1, Miz1 (Herold et al, 2002) and HPV16 E2 (Boner et al, 2002), suggesting that new functions of transcriptional regulation were acquired by metazoan TopBP1 during evolution. Miz1 mediates cell cycle arrest by transactivating p15INK4b (Seoane et al, 2001; Staller et al, 2001) and p21Cip1 (Herold et al, 2002; Seoane et al, 2002). TopBP1 interacts with and represses the transcriptional activities of Miz1. Thus, TopBP1 may have a more general role in transcriptional regulation. Here, we show that TopBP1 is regulated by the PI3K–Akt pathway. Phosphorylation of TopBP1 by Akt kinase induces TopBP1 oligomerization and binding to E2F1. Phosphorylation by Akt is also required for TopBP1 to interact with other transcription factors, such as HPV16 E2 and Miz1, and repress Miz1 activity. These results link PI3K–Akt survival signaling with the control of E2F1-induced apoptosis and Miz1-mediated cell cycle arrest through regulation of TopBP1 oligomerization. Results Akt phosphorylates TopBP1 in vitro and in vivo, and induces the interaction between TopBP1 and E2F1 As E2F1-induced apoptosis is inhibited by the Akt pathway (Hallstrom and Nevins, 2003), we speculated that TopBP1-mediated E2F1 regulation might be regulated by Akt. Upon examination of the TopBP1 sequence, we identified a single Ser1159 residue lying within an optimal surrounding sequence for Akt phosphorylation (RXRXXS/T). Moreover, this site and its surrounding sequences (RARLAS1159) are completely conserved among frog, mouse, rat and human TopBP1 (Figure 1A). Thus, we hypothesized that Akt might phosphorylate TopBP1 at this site. Indeed, purified Akt effectively phosphorylated GST-TopBP1, which was produced and purified from Escherichia coli, and the phosphorylation was completely abrogated by mutation of Ser1159 to Ala (S1159A) (Figure 1B). This result demonstrates that Akt can directly phosphorylate TopBP1 at Ser1159 in vitro. To test whether endogenous TopBP1 was phosphorylated in vivo, we utilized a monoclonal antibody (110B7) specifically recognizing phospho-(Ser/Thr) Akt substrates. This antibody can specifically recognize the phosphorylated Ser1159 peptide, but not its unphosphorylated counterpart (Supplementary Figure 1A). It also specifically recognized pSer1159-TopBP1 on immunoblotting (Figures 2E, 5C and 6H). The endogenous TopBP1 was immunoprecipitated from HEK293 cells with a TopBP1-specific antibody and immunoblotted with this phospho-Akt substrate-specific antibody. As shown in Figure 1C, TopBP1 was recognized by this phospho-specific antibody in an Akt-dependent manner. Endogenous Akt was at least partially active in growing HEK293 cells, as it could be recognized by a phospho-Ser473-specific Akt antibody, a marker of Akt activity (Figure 1C, lower panels). When transfected with a dominant-negative Akt (dn-Akt) or treated with a PI-3 kinase inhibitor LY294002, phosphorylation of TopBP1 was inhibited (Figure 1C). This result strongly suggests that TopBP1 is phosphorylated by Akt in vivo. We further tested the response to physiological Akt activators. Phosphorylation of TopBP1 was greatly induced by insulin and serum (Figure 1D and E). Both insulin and serum also stimulated the interaction between endogenous TopBP1 and E2F1. To confirm that Ser1159 was the phosphorylation site in vivo, we expressed TopBP1 or S1159A mutant in the presence of dn-Akt or constitutively active Akt (ca-Akt) in HEK293 cells and performed metabolic labeling with 32P-orthophosphate. Phosphorylation of wild-type TopBP1 was much stronger in the presence of ca-Akt compared with that in dn-Akt, and importantly, mutation of Ser1159 of TopBP1 to Ala blocked Akt-dependent phosphorylation (Figure 1F). Consistent with the interaction between TopBP1 and E2F1 during G1/S (Liu et al, 2004), phosphorylation of TopBP1 by Akt also peaks at G1/S transition (Supplementary Figure 1B). Together, these results indicate that Akt phosphorylates TopBP1 at Ser1159 in vitro and in vivo, and induces the interaction between TopBP1 and E2F1. Figure 1.Akt phosphorylates TopBP1 in vitro and in vivo, and induces the interaction between endogenous TopBP1 and E2F1. (A) The RXRXXS surrounding Ser1159 is conserved among human, mouse, rat and frog TopBP1. (B) Purified GST, GST-TopBP1 or GST-TopBP1(S1159A) protein was incubated with recombinant Akt kinase in the presence of [γ-32P]ATP. GST proteins were then pulled down with glutathione-Sepharose followed by SDS–PAGE. The upper panel depicts the 32P autoradiograph and the lower panel shows an immunoblot with GST antibody. (C) HEK293 cells were either transfected with dn-Akt or treated with LY294002 (25 μM for 24 h) and lysates were immunoprecipitated (IP) with TopBP1 antibody (αTopBP1) or normal mouse IgG and immunoblotted (IB). An aliquot of input lysates was subject to SDS–PAGE and immunoblotted with antibodies for Akt or phospho-Akt (Ser473) (lower panel). (D) HEK293 cells were starved in serum-free medium for 12 h and then treated with insulin (500 nM) for 10 and 60 min. The cell lysates were immunoprecipitated with TopBP1 antibody or normal mouse IgG and immunoblotted. An aliquot of input lysates was subjected to SDS–PAGE and immunoblotted with antibodies for E2F1, Akt or phospho-Akt (Ser473) (lower panel). (E) HEK293 cells were starved in serum-free medium for 12 h and then stimulated by 10% FBS for 120 min. Immunoprecipitation and immunoblotting were performed as panel D. (F) HEK293 cells were trasnfected with FLAG-tagged TopBP1 or S1159A mutant, and dn-Akt or ca-Akt, as indicated. The cells were then metabolically labeled with 32P-orthophosphate followed by anti-FLAG beads immunoprecipitation and autoradiograph (upper panel) or immunoblotting (lower panel). Download figure Download PowerPoint Figure 2.Akt phosphorylation is required for TopBP1 to interact with E2F1. (A) The membrane for endogenous TopBP1 immunoprecipitates shown in Figure 1C was striped and probed with an E2F1 antibody to detect the interaction between endogenous E2F1 and TopBP1. (B) HEK293 cells were transfected with E2F1 and FLAG-TopBP1 and treated with LY294002. The lysates were then immunoprecipitated with anti-FLAG beads followed by immunoblotting. The endogenous E2F1 was barely detectable in these films due to a relatively short exposure in detecting overexpressed proteins. (C) HEK293 cells were transfected with E2F1, FLAG-TopBP1 (either wild type, or S1159A or S1159D) and ca-Akt or dn-Akt, and the lysates were subjected to immunoprecipitation and immunoblotting as in panel A. (D) Akt phosphorylation induces the interaction between TopBP1 and E2F1 in vitro. Purified TopBP1(W: wild type) or TopBP1(A: S1159A) protein was first phosphorylated with Akt kinase and then incubated with purified GST or GST-E2F1. GST pulldown with glutathione-Sepharose was performed and probed with indicated antibodies. (E) The input TopBP1 protein after phosphorylation with Akt kinase in panel D was immunoblotted with phospho-Akt substrate antibody (110B7) or TopBP1 antibody. Download figure Download PowerPoint Figure 3.A physiological role of Akt phosphorylation of TopBP1 in the control of endogenous E2F1 target gene expression and E2F1 transcriptional activity. (A) NIH3T3 cells were transfected with a control siRNA vector (pSUPER-siGFP) or pSUPER-siTopBP1. Twenty-four hours later, the cells were infected with adenoviruses either harboring an empty vector or expressing TopBP1 or S1159A mutant TopBP1 at an m.o.i. of 400. Two days later, RNA was extracted and RT–PCR analysis was performed. Right panel: An aliquot of cell lysates was analyzed by Western blot with TopBP1 or β-actin antibody. (B) NIH3T3 cells were transfected with a control vector (pSUPER-siGFP) or pSUPER-siTopBP1, with or without coexpression of ca-Akt. Two days later, the cells were left untreated (no tx) or treated with adriamycin (Adr, 1 μM) for 16 h, and RT–PCR and Western blot analysis were performed as in panel A. (C) E2F1 activity was measured in HEK293 cells by p14ARF promoter-luciferase activity assay (Liu et al, 2003). The cells were either left untreated or treated with LY294002 for 18 h. Luciferase activity of transfected E2F1 was determined as fold induction relative to that of empty vector control. Each sample was performed in triplicate. The experiments were repeated multiple times with consistent results. One-tenth of each sample was subjected to Western blot analysis (upper panels, E: E2F1, T2: TopBP1 2 μg, T10: TopBP1 10 μg). (D) E2F1 activity was assayed in HEK293 cells with or without cotransfection of dn-Akt plasmid. (E) TopBP1, either wild type, S1159A or S1159D mutant, was cotransfected with E2F1 to REF52 fibroblasts. Some transfected cells were treated with LY294002 as in panel C. The E2F1 activity was then assayed. The expression levels of E2F1 and TopBP1 were determined by immunoblotting (upper panels). (F) The regulation of E2F1 by TopBP1 is independent of all pocket proteins. E2F1 transcriptional activity was assayed in wild-type (wt) MEFs or Rb−/−;p107−/−;p130−/− TKO MEFs. The activity of E2F1 in the presence of TopBP1 was determined as the percentage relative to that in the absence of TopBP1. An aliquot of each sample was subjected to E2F1 Western blot analysis (right panels, E: E2F1, T2: TopBP1 2 μg, T5: TopBP1 5 μg). The experiments were performed in two independent TKO MEFs with consistent results. Download figure Download PowerPoint Figure 4.Akt phosphorylation of TopBP1 is required for TopBP1 to repress E2F1-mediated apoptosis under physiological conditions. (A) NIH3T3 cells were transfected with a control siRNA vector (pSUPER-siScramble) or pSUPER-siTopBP1 along with GFP-expressing plasmid. Twenty-four hours later, the cells were infected with adenoviruses either harboring an empty vector or expressing TopBP1 or S1159A mutant TopBP1 at an m.o.i. of 400. Two days later, cells were stained with annexin V-PE and 7-AAD, and GFP-positive cells were gated and analyzed by flow cytometry. The data shown are the mean±s.e. of three independent experiments. An aliquot of cell lysates was analyzed by Western blot with TopBP1 and β-actin antibodies (upper panels). (*) P<0.001 (t-test) compared with siScramble; (**) P<0.01 (t-test) compared with wild-type TopBP1 rescue. (B) A representative profile of apoptosis as described in panel A. The percentage of annexin-positive cells is shown on top of each profile. (C) REF52 cells were serum-starved and infected with AdE2F1 with/without AdTopBP1 or its mutants at an m.o.i. of 100. The cells were then grown in 0.25% serum for 3 days and apoptosis was scored by annexin V-PE/7-AAD staining. Some AdE2F1-infected cells were grown in 10% serum after infection. The data shown are the mean±s.e. of four independent experiments. (*) P<0.05 (t-test) compared with AdE2F1; (**) P<0.05 (t-test) compared with AdE2F1. (D) A parallel experiment as in panel C was performed except that the cellular lysates were harvested 2 days after infection. Immunoblottings were performed to ensure equal expression of E2F1 and TopBP1 among different samples. Presence of the 89 kDa proteolytic fragment of PARP is indicative of apoptosis. Download figure Download PowerPoint Figure 5.Akt-dependent oligomerization of TopBP1. (A) HEK293 cells were transfected with FLAG-TopBP1 along with Myc-TopBP1 or Myc-TopBP1(S1159A) (Myc-S1159A) as well as Akt plasmids. The lysates were then immunoprecipitated with anti-FLAG beads followed by immunoblotting as indicated. The overexpression of TopBP1 and dn-Akt in the input lysates is shown in lower panels. The ca-Akt, HA-myr(Δ4–129) Akt, cannot be detected by this Akt antibody, which recognizes the N-terminus of Akt, but can be detected by HA antibody (Figure 2C and data not shown). Transfection of ca-Akt may lead to increasing expression of endogenous Akt in some experiments. (B) HEK293 cells were transfected with FLAG-BRCT678 along with Myc-TopBP1 or Myc-TopBP1(S1159A) (Myc-S1159A) as well as Akt plasmids. The immunoprecipitation by anti-FLAG beads was then carried out as in panel A. (C) HEK293 cells were transfected with FLAG-BRCT78 and Myc-BRCT78 or their S1159A mutant counterparts, or with dn-Akt. The lysates were then immunoprecipitated with anti-FLAG beads followed by immunoblotting. The expression of Myc-BRCT78 or S1159A mutant was confirmed by immunoprecipitation and immunoblotting with anti-Myc (middle panels). Phosphorylation of Ser1159 in Myc-BRCT78 was detected by phospho-Akt substrate antibody. Total lysates were also immunoblotted directly to detect the expression of HA-Akt (lower panel). (D) HEK293 cells were transfected with FLAG-TopBP1 and Myc-TopBP1 or FLAG-TopBP1Δ78 (FLAG-Δ78) and Myc-TopBP1Δ78 (Myc-Δ78). The lysates were then immunoprecipitated with anti-FLAG beads followed by immunoblotting with Myc antibody (upper panel) and FLAG antibody (middle panel) or immunoprecipitated with anti-Myc beads followed by immunoblotting with Myc antibody (lower panel). (E) Akt phosphorylation induces self-association of TopBP1 in vitro. Purified TopBP1 was incubated with GST-BRCT78. Some TopBP1 or GST-BRCT78 was phosphorylated with recombinant Akt kniase as indicated before incubation. S1159A mutant (A) of TopBP1 or BRCT78 serves as a control for its wild-type (wt) counterpart. GST pulldown with glutathione-Sepharose was then performed and probed with TopBP1 antibody to detect the interaction between full-length TopBP1 and BRCT78. GST-BRCT78 was visualized by Ponceau S staining. Download figure Download PowerPoint Figure 6.E2F1 binding and regulation require oligomerization of TopBP1. A TopBP1 dominant-negative mutant induces E2F1-dependent apoptosis. (A) E2F1 transcriptional activity was determined by p14ARF promoter-driven luciferase assay in the presence of TopBP1 and BRCT78 or BRCT78(S1159A) at different ratios. A TopBP1-expressing plasmid (10 μg) was cotransfected with plasmids expressing BRCT78 or BRCT78(S1159A) at 5 or 10 μg. E2F1 immunoblot from lysates of each transfection is shown on the top. (*) P<0.05 (t-test) compared with E2F1+TopBP1; (**) P<0.05 (t-test) compared with E2F1+TopBP1+BRCT78 (10 μg). (B) HEK293 cells were transfected with E2F1 plasmid along with TopBP1 or BRCT78 or TopBP1 and BRCT78 plasmids. The lysates were immunoprecipitated with anti-FLAG beads and immunoblotted with E2F1 antibody (upper panel) or TopBP1 antibody against the carboxyl terminus of TopBP1, which recognizes both TopBP1 and BRCT78 (middle panel). The input lysates were also immunoblotted with E2F1 antibody (lower panel). (C) HEK293 cells were transfected with the plasmid expressing TopBP1-BRCT78, or TopBP1-BRCT78(S1159A), and GFP along with pSUPER-siE2F1 or a control siRNA pSUPER-siScramble. Two days later, cells were stained with annexin V-PE and 7-AAD, and GFP-positive cells were gated and analyzed by flow cytometry. The data shown are the mean±s.e. of three independent experiments. An aliquot of cell lysates was analyzed by Western blot with BRCT78, E2F1 and β-actin antibodies. (*) P<0.001 (t-test) compared with pcDNA3+siScramble; (**) P<0.05 (t-test) compared with BRCT78+siScramble. (D) A representative profile of apoptosis as described in panel C. The percentage of annexin-positive cells is shown on top of each profile. (E) Wild-type and E2F1−/− MEFs were transfected with the plasmid expressing TopBP1-BRCT78, or TopBP1-BRCT78(S1159A), and GFP. Apoptosis was analyzed as described in panel C. The data shown are the mean±s.e. of three independent transfections. An aliquot of cell lysates was analyzed by Western blot with BRCT78, E2F1 and β-actin antibodies (upper panels). (*) P<0.05 (t-test) compared with pcDNA3 vector control; (**) P<0.05 (t-test) compared with BRCT78. (F) A representative profile of apoptosis as described in panel E. The percentage of annexin-positive cells is shown on top of each profile. (G) Sequence alignment of TopBP1 orthologs from human, mouse, rat and frog, and other BRCT repeats-containing proteins. The boxed sequences of BRCA1 represent the residues that form hydrogen bonds with pSer990 of BACH1 (Shiozaki et al, 2004). (H) GST-BRCT78 was produced in E. coli and purified with glutathione-Sepharose. The GST portion was removed by PreScission protease. Purified BRCT78 was incubated with Btn-pS1159 peptide (pP) or Btn-nonphosophorylated peptide (nP) followed by streptavidin Sepharose pulldown and immunoblotting with anti-TopBP1 carboxyl-terminus antibody. To ensure the loading of peptides, the input peptides were separated in 20% SDS–PAGE and immunoblotted with p-Akt substrate antibody or visualized by Coomassie blue staining. Download figure Download PowerPoint Akt phosphorylation is required for TopBP1 to bind E2F1 and regulate E2F1 activity Next we investigated the role of Akt phosphorylation in the interaction between TopBP1 and E2F1. The interaction between endogenous E2F1 and TopBP1 was inhibited by LY294002 or dn-Akt, as assayed by immunoprecipitation (Figure 2A). The interaction between transfected E2F1 and TopBP1 was also inhibited by LY294002 treatment or dn-Akt (Figure 2B and C). The S1159A mutant failed to interact with E2F1 even in the presence of ca-Akt (Figure 2C). The S1159D mutant of TopBP1 that mimics the phosphorylated form was able to interact with E2F1, although to a lesser extent when compared with wild type. However, its binding was no longer inhibited by the expression of dn-Akt (Figure 2C). The interaction of purified proteins was also tested in a cell-free system. Phosphorylation of TopBP1 at Ser1159 by a recombinant Akt kinase greatly induced its interaction with E2F1 in vitro (Figure 2D and E). Based on these results, we conclude that phosphorylation of TopBP1 by Akt at Ser1159 is required for TopBP1 to bind E2F1. We then investigated whether Akt phosphorylation was required for TopBP1 to repress E2F1 activity by examining the endogenous E2F1 target genes. Previously, we showed that knockdown of TopBP1 by small interfering RNAs (siRNAs) de-repressed E2F1 target genes in T98G cells (Liu et al, 2004). TopBP1 knockdown in NIH3T3 cells by siRNA against murine TopBP1 also de-repressed E2F1 target genes such as p73, Apaf-1, caspase 3, cyclin E, thymidine kinase 1 (TK) and p107 (Figure 3A). The expression of TopBP1 was then reconstituted with wild-type or S1159A TopBP1 by recombinant adenoviruses that express human TopBP1 transcripts resistant to the siRNA against murine TopBP1 due to codon degeneracy. Importantly, reconstitution of wild-type TopBP1 but not S1159A mutant to physiological levels re-suppressed the expression of E2F1 target genes (Figure 3A). To further test the model that Akt controls E2F1 through TopBP1, we examined whether activated Akt could suppress the expression of E2F1 target genes after genotoxic stress and whether this effect depended on TopBP1. As shown in Figure 3B, the expressions of p73, Apaf-1, caspase 3, cyclin E, TK and p107 were induced by adriamycin, and expression of ca-Akt suppressed these gene expressions in a TopBP1-dependent manner. We cannot rule out the possibility that some of the effect from expression of ca-Akt may be p53-dependent; however, given the established role of E2F1 in controlling the expression of these target genes, particularly cyclin E, TK and p107, the observed effect most likely reflects E2F activity. Interestingly, the levels of cyclin D1 (Figure 3A and B) transcripts were not significantly changed by TopBP1 knockdown or Akt activation. As the cyclin D1 promoter is also directly regulated by E2F1 (Ohtani et al, 1995; Lee et al, 2000), this result suggests that TopBP1 may specifically regulate a subset of E2F1 target genes. The regulation of E2F1 transcriptional activity by TopBP1 was also demonstrated by an E2F1 reporter assay. The ability of TopBP1 to repress E2F1 activity was blocked by LY294002 (Figure 3C) and dn-Akt (Figure 3D). Mutation of Ser1159 to Ala also blocked TopBP1's ability to repress E2F1 in both REF52 fibroblasts (Figure 3E) and HEK293 cells (Supplementary Figure 2). Consistent with the findings in co-immunoprecipitation experiments (Figure 2C), S1159D mutant also repressed E2F1, although to a lesser extent; nevertheless, its repressive effect was not significantly inhibited by LY294002 (Figure 3E). Taken together, we conclude that Akt phosphorylates TopBP1 at Ser1159 to repress E2F1 transcriptional activity. Regulation of E2F1 by TopBP1 is independent of all pocket proteins Previous work showed that TopBP1 could repress E2F1 in Rb-deficient cells (Saos-2 and Rb−/− mouse embryonic fibroblasts (MEFs)) (Liu et al, 2004). To rule out the possibility that the other pocket proteins p107 and p130 may be involved, we examined the effect of TopBP1 on E2F1 transcriptional activity in Rb−/−;p107−/−;p130−/− triple knockout (TKO) MEFs (Sage et al, 2000). TopBP1 effectively repressed E2F1 activity both in wild-type and TKO MEFs (Figure 3F). Thus, the regulation of E2F1 by TopBP1 is distinct from the classic Rb family/E2F control. Akt phosphorylation of TopBP1 is required for TopBP1 to repress E2F1-mediated apoptosis We also examined the ability of TopBP1 mutants in repressing E2F1-induced apoptosis. Prior studies have shown that TopBP1 siRNA induced E2F1-dependent apoptosis in HEK293 cells and MEFs (Liu et al, 2004). TopBP1 knockdown in NIH3T3 cells also induced apoptosis, which was suppressed by reconstitution of wild-type TopBP1 expression (Figure 4A and B). Importantly, reconstitution with S1159A-TopBP1 failed to repress the apoptosis. This result is consistent with the examination of E2F1 target genes (Figure 3A), and strongly argues for a physiological role of Akt phosphorylation in the control of E2F1 proapoptotic function. The requirement of Akt phosphorylation for TopBP1 to control E2F1-induced apoptosis was further demonstrated by an independent assay. Infection of recombinant adenoviruses expressing E2F1 in serum-starved REF52 cells induces apoptosis, which is inhibited by coexpression of TopBP1 (Liu et al, 2003). We infected serum-starved REF52 cells with recombinant adenoviruses expressing E2F1 (AdE2F1) and either TopBP1 (AdTopBP1) or its mutants, and assessed apoptosis by annexin staining (Figure 4C and Supplementary Figure 3). Expression of E2F1 induced apoptosis in the absence of serum, which was largely abrogated by addition o