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Evidence for substrate control of Cdk phosphorylation timing during the budding yeast cell cycle 2522

ABSTRACT: The main intention of this project is to investigate how Cdk substrate phosphorylation is ordered during the cell cycle. We want to understand what makes a ‘good’ Cdk substrate that is phosphorylated early during the cell cycle, e. g. to promote S phase, as compared to a ‘bad’ Cdk substrate that is phosphorylated much later, e. g. when cells enter mitosis. This could be because a late substrate is indeed a poor Cdk substrate, or it could be that the late substrate is a good substrate for Cdk counteracting phosphatases. The two possibilities are not mutually exclusive. We want to compare unperturbed phosphorylation and rephosphorylation from an artificially low phosphorylation state in metaphase arrest. To do that, we arrest cells in metaphase, inhibit Cdk until substrates get dephosphorylated, and reactivate Cdk and record the time and kinetics of the substrate rephosphorylation.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Helen Flynn

LAB HEAD: Frank Uhlmann

PROVIDER: PXD054974 | Pride | 2025-04-10

REPOSITORIES: Pride

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Project description:This model is from the article: Downregulation of PP2A(Cdc55) phosphatase by separase initiates mitotic exit in budding yeast. Queralt E, Lehane C, Novak B, Uhlmann F. Cell. 2006 May 19;125(4):719-32. 16713564 , Abstract: After anaphase, the high mitotic cyclin-dependent kinase (Cdk) activity is downregulated to promote exit from mitosis. To this end, in the budding yeast S. cerevisiae, the Cdk counteracting phosphatase Cdc14 is activated. In metaphase, Cdc14 is kept inactive in the nucleolus by its inhibitor Net1. During anaphase, Cdk- and Polo-dependent phosphorylation of Net1 is thought to release active Cdc14. How Net1 is phosphorylated specifically in anaphase, when mitotic kinase activity starts to decline, has remained unexplained. Here, we show that PP2A(Cdc55) phosphatase keeps Net1 underphosphorylated in metaphase. The sister chromatid-separating protease separase, activated at anaphase onset, interacts with and downregulates PP2A(Cdc55), thereby facilitating Cdk-dependent Net1 phosphorylation. PP2A(Cdc55) downregulation also promotes phosphorylation of Bfa1, contributing to activation of the "mitotic exit network" that sustains Cdc14 as Cdk activity declines. These findings allow us to present a new quantitative model for mitotic exit in budding yeast. This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2012 The BioModels.net Team. For more information see the terms of use . To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.

			Action	DRS
	ED2522_LC_txt.zip	Other
	ED2522_MC1_txt.zip	Other
	ED2522_MC2_txt.zip	Other
	ED2522_SMOAC_txt.zip	Other
	ED2522_takacsl_FeNTA_HCD.raw	Raw

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Evidence for substrate control of Cdk phosphorylation timing during the budding yeast cell cycle 2522

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