1. A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
- Author
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Verena M. Link, David Gonzalez, John D. Lapek, Yeon J. Lee, Christopher K. Glass, Manuel Kaulich, and Steven F. Dowdy
- Subjects
Checkpoints ,Cyclin E ,Proteome ,Cell division ,Cells ,1.1 Normal biological development and functioning ,Science ,Cyclin D ,Cell ,Cell Cycle Proteins ,Retinoblastoma Protein ,environment and public health ,Article ,03 medical and health sciences ,0302 clinical medicine ,Underpinning research ,Proto-Oncogene Proteins ,medicine ,Humans ,Phosphorylation ,Kinase activity ,Cells, Cultured ,030304 developmental biology ,Oncogene Proteins ,0303 health sciences ,Cultured ,Multidisciplinary ,biology ,Chemistry ,Cyclin-dependent kinase 2 ,G1 Phase ,Cyclin-Dependent Kinase 4 ,Cyclin-Dependent Kinase 6 ,Cell cycle ,Cell biology ,enzymes and coenzymes (carbohydrates) ,medicine.anatomical_structure ,biology.protein ,Medicine ,Generic health relevance ,biological phenomena, cell phenomena, and immunity ,mCherry ,Cell Division ,030217 neurology & neurosurgery - Abstract
During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.
- Published
- 2021
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