A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition

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:...

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Autores principales:	Manuel Kaulich, Verena M. Link, John D. Lapek, Yeon J. Lee, Christopher K. Glass, David J. Gonzalez, Steven F. Dowdy
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d7d64e91293648eb929fa5c8632d7b65
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spelling	oai:doaj.org-article:d7d64e91293648eb929fa5c8632d7b652021-12-02T16:17:17ZA Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition10.1038/s41598-021-94200-w2045-2322https://doaj.org/article/d7d64e91293648eb929fa5c8632d7b652021-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-94200-whttps://doaj.org/toc/2045-2322Abstract 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.Manuel KaulichVerena M. LinkJohn D. LapekYeon J. LeeChristopher K. GlassDavid J. GonzalezSteven F. DowdyNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Manuel Kaulich Verena M. Link John D. Lapek Yeon J. Lee Christopher K. Glass David J. Gonzalez Steven F. Dowdy A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
description	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.
format	article
author	Manuel Kaulich Verena M. Link John D. Lapek Yeon J. Lee Christopher K. Glass David J. Gonzalez Steven F. Dowdy
author_facet	Manuel Kaulich Verena M. Link John D. Lapek Yeon J. Lee Christopher K. Glass David J. Gonzalez Steven F. Dowdy
author_sort	Manuel Kaulich
title	A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
title_short	A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
title_full	A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
title_fullStr	A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
title_full_unstemmed	A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition
title_sort	cdk4/6-dependent phosphorylation gradient regulates the early to late g1 phase transition
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/d7d64e91293648eb929fa5c8632d7b65
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A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition

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