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Evidence for substrate control of Cdk phosphorylation timing during the budding yeast cell cycle 3270

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. As a reference dataset, I would like to know when the different proteins (and phosphosites) get phosphorylated as the cells go from G1 to metaphase during undisturbed cell cycle progression. This will allow me to compare phosphorylation kinetics with phosphorylation timing.

INSTRUMENT(S): Orbitrap Eclipse

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

SUBMITTER: Helen Flynn

LAB HEAD: Frank Uhlmann

PROVIDER: PXD054772 | Pride | 2025-04-10

REPOSITORIES: Pride

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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
	ED3270_LC_txt.zip	Other
	ED3270_MC1_txt.zip	Other
	ED3270_MC2_MS2_txt.zip	Other
	ED3270_MC2_MS3_txt.zip	Other
	ED3270_SMOAC_txt.zip	Other

Dataset Information

Evidence for substrate control of Cdk phosphorylation timing during the budding yeast cell cycle 3270

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