A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
- 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.
Author: | Manuel KaulichORCiD, Verena Maria KnieGND, John D. Lapek Jr, Yeon J. Lee, Christopher K. GlassORCiD, David J. Gonzalez, Steven F. DowdyORCiD |
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URN: | urn:nbn:de:hebis:30:3-632767 |
DOI: | https://doi.org/10.1038/s41598-021-94200-w |
ISSN: | 2045-2322 |
Parent Title (English): | Scientific reports |
Publisher: | Macmillan Publishers Limited, part of Springer Nature |
Place of publication: | [London] |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2021/07/19 |
Date of first Publication: | 2021/07/19 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2023/06/28 |
Tag: | Cell division; Checkpoints |
Volume: | 11 |
Issue: | art. 14736 |
Article Number: | 14736 |
Page Number: | 13 |
First Page: | 1 |
Last Page: | 13 |
Note: | Supplementary Information: The online version contains further supplementary material available at https://doi.org/10.1038/s41598-021-94200-w |
HeBIS-PPN: | 510041698 |
Institutes: | Medizin / Medizin |
Dewey Decimal Classification: | 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - CC BY - Namensnennung 4.0 International |