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• W2882997462 abstract "The Anaphase-Promoting Complex/Cyclosome (APC/C) drives degradation of a large variety of mitotic cell cycle regulators. Therefore, tight regulation of APC/C activity plays a central role in proper cell cycle progression. Suppression of APC/C activity during G2-phase is crucial for entry into mitosis. This is achieved 1) by Cdk-dependent phosphorylation of the APC/C activator subunit Fzr, which renders Fzr unable to interact with the APC/C, and 2) by direct inhibition of APC/C-Fzr by Regulator of Cyclin A1 (Rca1). Previous studies suggest that not only the C-terminal part, but also the central localized F-box in Rca1 is fundamental for APC/C-Fzr inhibition. F-box proteins usually recruit substrates to SCF complexes, causing their polyubiquitination and subsequent degradation. To identify such substrates, an Rca1 precipitate from S2R+ cells was analyzed by mass spectrometry. Rca1 was found in complex with SCF components and other cell cycle regulators including Dacapo (Dap) and Skp2. Rca1 binds polyubiquitinated substrates in an F-box dependent manner in vivo giving evidence for a functional SCF-Rca1 complex. To analyze, whether SCF-Rca1 affects the protein stability of Dap and Skp2, a method for the stability analysis of GFP-labeled proteins during cell cycle progression of S2R+ cells was established. Dap stability was analyzed using a cell cycle inactive GFP-tagged version of Dap that lacks destabilization by CRL4-Cdt2 during S-phase (GFP-Dap-dCDI-dPIPa). Downregulation of Rca1 activity by RNAi resulted in upregulation of GFP-Dap-dCDI-dPIPa protein levels. Conversely, overexpression of Rca1 stimulated degradation of GFP-Dap-dCDI-dPIPa in an F-box dependent manner. Overexpression of CycE enhanced the interaction between Rca1 and Dap-dCDI-dPIPa, and further destabilized GFP-Dap-dCDI-dPIPa. These studies indicate that SCF-Rca1 functions synergistically with CycE/Cdk2 to target Dap for degradation. Two candidate Cdk phosphorylation sites in Dap, S205 and S214, were mutated but these mutations did neither affect the stability nor did they impair the interaction with Rca1. Interaction analyses with different truncated Rca1 and Dap constructs have revealed that the N-terminal part of Dap and the central region in Rca1 are required for their interaction. Loss of Rca1 binding by deletion of N-terminal residues in GFP-Dap-dCDI-dPIPa resulted in its complete stabilization. Flow cytometric based in vivo APC/C-Fzr activity assays in S2R+ cells were used to analyze the functional interaction between SCF-Rca1 and Dap. Loss of the F-box reduced APC/C-Fzr inhibition. However, this defect in APC/C-Fzr inhibition was suppressed by RNAi-mediated downregulation of Dap activity. Taken together, these studies provide a model, in which SCF-Rca1 targets Dap for degradation to release CycE/Cdk2 activity for APC/C-Fzr inhibition. To analyze the biochemical inhibition of APC/C-Fzr by Rca1 in more detail, an in vitro APC/C-Fzr ubiquitination assay was further developed. Experiments in the past failed to purify active Drosophila APC/C-Fzr from transgenic embryos. Therefore, a stable S2R+ cell line expressing a GFP-tagged APC/C component, Cdc16-MYC-TEV-GFP, was established. In addition, Fzr was in vitro translated in reticulocyte lysate and purified from baculovirus-infected SF-21 cells. APC/C-Fzr-dependent ubiquitination activity was detected to some extend by using Cdc16 precipitate treated with in vitro translated Fzr." @default.
• W2882997462 created "2018-08-03" @default.
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• W2882997462 date "2018-07-13" @default.
• W2882997462 modified "2023-09-27" @default.
• W2882997462 title "Dual regulation of APC/C activity by Rca1" @default.
• W2882997462 hasPublicationYear "2018" @default.
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