Collaborative role of E2F transcriptional activity and G1 cyclindependent kinase activity in the induction of S phase - PubMed (original) (raw)
Collaborative role of E2F transcriptional activity and G1 cyclindependent kinase activity in the induction of S phase
G Leone et al. Proc Natl Acad Sci U S A. 1999.
Abstract
A considerable body of evidence points to a role for both cyclin E/cyclin-dependent kinase (cdk)2 activity and E2F transcription activity in the induction of S phase. We show that overexpression of cyclin E/cdk2 in quiescent cells induces S phase, that this coincides with an induction of E2F activity, and that coexpression of E2F enhances the cyclin E/cdk2-mediated induction of S phase. Likewise, E2F overexpression can induce S phase and does so in the apparent absence of cyclin E/cdk2 activity. In addition, although the inhibition of cyclin E/cdk2 activity blocks the induction of S phase after growth stimulation of normal mouse embryo fibroblasts, inhibition of cyclin E/cdk2 does not block S phase induction in Rb-/- cells where E2F activity is deregulated. These results point to the important roles for E2F and cyclin E/cdk2 in the induction of S phase. Moreover, the nature of the E2F targets and the suspected targets for cyclin E/cdk2 suggests a potential molecular mechanism for the collaborative action of cyclin E/cdk2 and E2F in the induction of S phase.
Figures
Figure 1
E2F and cyclin E/cdk2 collaborate in S phase induction. Analysis of S phase induction with flow cytometry. Cells were starved and infected with Ad-Con (control, moi = 600 ffu per cell), Ad-E2F1 (moi = 100 ffu per cell), Ad-E2F2 (moi = 100 ffu per cell), and Ad-CycE and Ad-Cdk2 (moi = 200 ffu per cell and 300 ffu per cell, respectively) as indicated; the final moi for each sample was adjusted to 600 ffu per cell with Ad-Con. Cells were then incubated with fresh starvation medium or with medium containing 10% serum (+) and then harvested at the indicated times and processed for flow cytometry. The horizontal axis reflects relative DNA content, and the vertical axis represents cell number.
Figure 2
Overexpression of cyclin E/cdk2 induces Rb phosphorylation and E2F-dependent transcription. (A) Assay for Rb phosphorylation. Cells treated as in Fig. 1 were harvested in SDS/PAGE loading buffer at 24 hr postinfection, and Rb (Endog. Rb-P) was detected by using Western blotting (Upper). Alternatively, cells treated as in Fig. 1 were lysed in immunoprecipitation buffer, and the lysates were used for immunoprecipitation with antibodies specific for cyclin E, and the cyclin E-associated kinase assays were performed as described (42) by using histone H1 as a substrate (Lower). (B) E2F activity measured by using Northern analysis. Cells treated as in A were harvested at 24 hr postinfection and processed for Northern analysis by using specific probes for proliferating cell nuclear antigen (PCNA), B-Myb, and glyceraldehyde-3-phosphate dehydrogenase.
Figure 3
E2F1 induces S phase in the absence of cdk activity. (A) E2F-mediated S phase induction in p21-arrested cells. REF52 cells were held at density arrest for 24 hr and infected with either Ad-Con (control virus with empty expression cassette) at a moi of 125 ffu per cell, Ad-E2F1 or Ad-E2F1/VP16 (AVP16′′) at an moi of 25 ffu per cell together with Ad-p21 (moi of 50, 100, or 200 ffu per cell as indicated) or Ad-Np21 (moi of 100 ffu per cell), and then replated at subconfluent densities. Cells were labeled with BrdUrd from 12 to 24 hr postinfection, and BrdUrd was detected by using indirect immunofluorescence. At least 300 nuclei were scored for BrdUrd immunofluorescence. (B) E2F induces DNA replication. REF52 cells were held at density arrest for 24 hr before infection with either Ad-Con (moi of 125 ffu per cell), Ad-p21 (moi of 100 ffu per cell), or Ad-p21 and Ad-E2F1 or Ad-E2F2 (moi of 100 and 25 ffu per cell, respectively). Cells were then replated at subconfluent densities, harvested at the indicated times postinfection, and processed for flow cytometry. The horizontal axis reflects relative DNA content and the vertical axis represents cell number. (C) E2F-mediated S phase induction in the absence of cyclin D- or cyclin E-associated kinase activity. Cells treated as in A were lysed in IP buffer, and the lysates were immunoprecipitated with control IgG antibodies (Ig) or antibodies against either cyclin D1 or cyclin E. The sample designated A represents lysates made from density-arrested cells immunoprecipitated with the corresponding specific antibodies. The kinase assays were performed as described (42) except glutathione _S_-transferase–Rb was used as the substrate. (D) E2F-mediated S phase induction in the absence of cdk2- or cyclin A-associated kinase activity. Cells treated as in A were lysed in IP buffer and the lysates immunoprecipitated with control IgG antibodies (Ig) or antibodies against either cdk2, cyclin A, or cyclin B1. The samples designated A represent lysates made from density-arrested cells immunoprecipitated with the corresponding specific antibodies. The kinase assays were performed as in C. (E) E2F-mediated S phase and cyclin B1-associated kinase induction. Density-arrested REF52 cells were infected with either Ad-Con (moi of 125 ffu per cell; open bar), Ad-p21 (moi of 100 ffu per cell, gray bar), or Ad-p21 and Ad-E2F1 (moi of 100 ffu per cell and 125 ffu per cell, respectively, black bar) and replated at subconfluent densities. Cells were either labeled with BrdUrd for 1 hr before fixation at the indicated times and processed as in A (Upper) or lysed in IP buffer at the indicated times and assayed for cyclin B1-associated kinase activity as in D (Lower). (F) Assay for Rb phosphorylation. Representative cell samples treated as in A were harvested in SDS/PAGE loading buffer, and Rb was detected by using Western blotting. To better visualize the Rb product in lane 2, we show a 3-fold longer exposure of this sample in lane 1.
Figure 4
E2F overexpression induces Mcm protein accumulation. REF52 cells were starved and infected with Ad-Con (moi of 100 ffu per cell) or Ad-E2F2 (moi of 100 ffu per cell) as described. Cells were then incubated with fresh starvation medium or with medium containing 10% serum (control +) and then harvested 24 hr postinfection. Endogenous Mcm2 and Mcm3 proteins were detected by using Western blot analysis with antibodies specific for each protein.
Figure 5
Inhibition of cyclin E/cdk2 does not block S phase in the absence of Rb. (A) Inhibition of S phase induction by p21. Mouse embryo fibroblasts (MEF) from wild-type (Rb+/+) or knockout (Rb−/−) embryos were held at density arrest for 24 hr and infected with either Ad-Con (control virus with empty expression cassette) at a moi of 125 ffu per cell or Ad-p21 (moi of 100 or 500 ffu per cell as indicated) and then replated at subconfluent densities. Cells were labeled with BrdUrd from 12 to 24 hr postinfection, and BrdUrd was detected by using indirect immunofluorescence. At least 300 nuclei were scored for BrdUrd immunofluorescence. (B) Inhibition of cdk activity by p21. Cells treated as in A or cells density-arrested for 24 hr were lysed in IP buffer and immunoprecipitated with antibodies against either cyclin E, cyclin A, or cdk2. The kinase assays were performed by using glutathione _S_-transferase–Rb as the substrate.
Figure 6
Collaboration of cyclin E/cdk2 with E2F in the induction of S phase. The figure depicts the interaction of the multisubunit complex known as ORC with a DNA replication origin. In addition to the Orc1 protein, the accumulation of additional components of the initiation complex, including each of the Mcms and Cdc6, depends on E2F activity. It is proposed that either the assembly of these components into a functional complex or the activation of an assembled complex is facilitated by cyclin E/cdk2-mediated phosphorylation, thereby triggering origin firing and the initiation of DNA replication.
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