TY  - JOUR
A1  - Kaulich, Manuel
A1  - Knie, Verena Maria
A1  - Lapek Jr, John D.
A1  - Lee, Yeon J.
A1  - Glass, Christopher K.
A1  - Gonzalez, David J.
A1  - Dowdy, Steven F.
T1  - A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
T2  - Scientific reports
N2  - 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.
KW  - Cell division
KW  - Checkpoints
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/63276
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-632767
SN  - 2045-2322
N1  - Supplementary Information: The online version contains further supplementary material available at https://doi.org/10.1038/s41598-021-94200-w
VL  - 11
IS  - art. 14736
SP  - 1
EP  - 13
PB  - Macmillan Publishers Limited, part of Springer Nature
CY  - [London]
ER  -