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:...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2021-07-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-94200-w |
| _version_ | 1818430466703753216 |
|---|---|
| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T15:33:52Z |
| format | Article |
| id | doaj.art-d7d64e91293648eb929fa5c8632d7b65 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T15:33:52Z |
| publishDate | 2021-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-d7d64e91293648eb929fa5c8632d7b652022-12-21T22:55:48ZengNature PortfolioScientific Reports2045-23222021-07-0111111310.1038/s41598-021-94200-wA Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transitionManuel Kaulich0Verena M. Link1John D. Lapek2Yeon J. Lee3Christopher K. Glass4David J. Gonzalez5Steven F. Dowdy6Department of Cellular and Molecular Medicine, University of California San DiegoDepartment of Cellular and Molecular Medicine, University of California San DiegoDepartment of Pharmacology, University of California San DiegoDepartment of Cellular and Molecular Medicine, University of California San DiegoDepartment of Cellular and Molecular Medicine, University of California San DiegoDepartment of Pharmacology, University of California San DiegoDepartment of Cellular and Molecular Medicine, University of California San DiegoAbstract 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.https://doi.org/10.1038/s41598-021-94200-w |
| spellingShingle | 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 Scientific Reports |
| title | 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_short | 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 |
| url | https://doi.org/10.1038/s41598-021-94200-w |
| work_keys_str_mv | AT manuelkaulich acdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT verenamlink acdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT johndlapek acdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT yeonjlee acdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT christopherkglass acdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT davidjgonzalez acdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT stevenfdowdy acdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT manuelkaulich cdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT verenamlink cdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT johndlapek cdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT yeonjlee cdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT christopherkglass cdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT davidjgonzalez cdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition AT stevenfdowdy cdk46dependentphosphorylationgradientregulatestheearlytolateg1phasetransition |