Cell Cycle Profiling Reveals Protein Oscillation, Phosphorylation, and Localization Dynamics

Abstract

The cell cycle is a highly conserved process involving the coordinated separation of a single cell into two daughter cells. To relate transcriptional regulation across the cell cycle with oscillatory changes in protein abundance and activity, we carried out a proteome- and phospho-proteome-wide mass spectrometry profiling. We compared protein dynamics with gene transcription, revealing many transcriptionally regulated G2 mRNAs that only produce a protein shift after mitosis. Integration of CRISPR/Cas9 survivability studies further highlighted proteins essential for cell viability. Analyzing the dynamics of phosphorylation events and protein solubility dynamics over the cell cycle, we characterize predicted phospho-peptide motif distributions and predict cell cycle-dependent translocating proteins, as exemplified by the S-adenosylmethionine synthase MAT2A. Our study implicates this enzyme in translocating to the nucleus after the G1/S-checkpoint, which enables epigenetic histone methylation maintenance during DNA replication. Taken together, this data set provides a unique integrated resource with novel insights on cell cycle dynamics. The cell cycle is a highly conserved process involving the coordinated separation of a single cell into two daughter cells. To relate transcriptional regulation across the cell cycle with oscillatory changes in protein abundance and activity, we carried out a proteome- and phospho-proteome-wide mass spectrometry profiling. We compared protein dynamics with gene transcription, revealing many transcriptionally regulated G2 mRNAs that only produce a protein shift after mitosis. Integration of CRISPR/Cas9 survivability studies further highlighted proteins essential for cell viability. Analyzing the dynamics of phosphorylation events and protein solubility dynamics over the cell cycle, we characterize predicted phospho-peptide motif distributions and predict cell cycle-dependent translocating proteins, as exemplified by the S-adenosylmethionine synthase MAT2A. Our study implicates this enzyme in translocating to the nucleus after the G1/S-checkpoint, which enables epigenetic histone methylation maintenance during DNA replication. Taken together, this data set provides a unique integrated resource with novel insights on cell cycle dynamics. The mechanism of cell division has been extensively studied for many decades resulting in a very detailed picture of the genes and proteins involved and their temporal function within the dividing cell. Historically, the cell cycle is divided into a DNA synthesis phase (S-phase) and a cell division phase (Mitosis; M-phase), with these two phases separated by two gap phases, G1 and G2. To date, many large-scale studies addressing the cell cycle focus on the transcriptional control of cell cycle regulated genes. Transcription can be used as a proxy for protein abundance and transcript dynamics translated to protein abundance on a larger scale for systems at the steady state (1Edfors F. Danielsson F. Hallström B.M. Käll L. Lundberg E. Pontén F. Forsström B. Uhlén M. Gene-specific correlation of RNA and protein levels in human cells and tissues.Mol. Syst. Biol. 2016; 12: 883Crossref PubMed Scopus (196) Google Scholar), but extrapolation between different mRNA-protein pairs has very little explanatory power (2Vogel C. Marcotte E.M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.Nat. Rev. Genet. 2012; 13: 227-232Crossref PubMed Scopus (2099) Google Scholar). Aside from translational control, allowing for regulation of protein turnover that is not reflected in mRNA levels, regulation by the Ubiquitin-Proteasome-System (UPS) 1The abbreviations used are:UPSUbiquitin-Proteasome-SystemPTMpost-translational modificationsFACSfluorescence-activated cell sorting. 1The abbreviations used are:UPSUbiquitin-Proteasome-SystemPTMpost-translational modificationsFACSfluorescence-activated cell sorting. can also influence protein abundance, causing a rapid decline in protein levels through ubiquitin-mediated degradation, an effect that is not reflected by mRNA levels. In addition, many cellular functions are regulated not by protein abundance at all, but rather by protein activation through post-translational modifications (PTMs), such as phosphorylation, or differential localization within the cell (3Orre, L. M., M., Vesterlund, Y., Pan, T., Arslan, Y., Zhu, A., Fernandes Woodbridge, O., Frings, E., Fredlund, and J., Lehtiö, . SubCellBarCode: Proteome-wide mapping of protein localization and relocalization. Mol. Cell.Google Scholar). Ubiquitin-Proteasome-System post-translational modifications fluorescence-activated cell sorting. Ubiquitin-Proteasome-System post-translational modifications fluorescence-activated cell sorting. Proteomic and transcriptomic analyses of cell cycle have previously been reported, often using chemical synchronization of cells. The disadvantage of synchronization-based cell-cycle analysis is the disruption of the natural cell cycle. Studies investigating cell cycle proteomics on asynchronous cells has been performed by Lamond and colleagues. In 2014 they used centrifugal elutriation to separate fractions enriched for cell cycle phases. They identified 358 cell-cycle dependent proteins, of which 31 also had cell-cycle dependent mRNA (4Ly T. Ahmad Y. Shlien A. Soroka D. Mills A. Emanuele M.J. Stratton M.R. Lamond A.I. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells.Elife. 2014; 3Crossref PubMed Scopus (80) Google Scholar). In a 2017 follow-up study, they segmented cells based on DAPI- and phospho-histone-H3 antibody staining on paraformaldehyde-fixated cells. This study included a phospho-proteomic analysis, and a further focus on mitotic T. A. B. D. Lundberg E. Lamond A.I. Proteomic analysis of cell cycle in asynchronous mitotic using Scopus Google Scholar). We to further the and process of the cell cycle at the protein in asynchronous cells. studies that on chemical synchronization of cells or centrifugal (4Ly T. Ahmad Y. Shlien A. Soroka D. Mills A. Emanuele M.J. Stratton M.R. Lamond A.I. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells.Elife. 2014; 3Crossref PubMed Scopus (80) Google C. A of mitotic PubMed Scopus Google M. A. C. F. 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Hallström B.M. Käll L. Lundberg E. Pontén F. Forsström B. Uhlén M. Gene-specific correlation of RNA and protein levels in human cells and tissues.Mol. Syst. Biol. 2016; 12: 883Crossref PubMed Scopus (196) Google Scholar). for the temporal dynamics of the cell cycle, the between and is the cell an mRNA in cell cycle phase in for protein in the during the transcriptional of mitosis. This the to the cell cycle of proteins using such as or mass rather on The DNA is not cell cycle using gene analysis, to be by the in cells The that this a separation of function between and the DNA which during C. F. M. cycle regulation as a mechanism for separation of the DNA and PubMed Scopus Google Scholar). not in data we can the G1 of and is in and phases. also to DNA the that is also in gene expression Y. and the dynamics of DNA PubMed Scopus Google Scholar). as a novel for with through the transcriptional regulation of genes for cell cycle and L. Y. E. M. A. A. L. A. DNA as a novel for PubMed Scopus Google Scholar). the of protein we a of essential proteins in the and this an to protein levels in that essential proteins which are in are to be and not be identified as essential in CRISPR/Cas9 survivability of these cell cycle which only a in out of cell in T. A. E. B. Y. M. A CRISPR/Cas9 and of the human Biol. PubMed Scopus Google Scholar), cell cycle-dependent phosphorylation and transcriptional protein levels the the phases We that this is of a of and the of cells the transcriptomic of is that M. A. E. D. F. L. A. M. M. D. The cell 2016; PubMed Scopus Google Scholar), in cells by that is in and in G1 M. C. C. D. is a rather a of PubMed Scopus Google Scholar), and is a process for of which within at the of A. T. A. T. T. M. A. dynamics of cell-cycle PubMed Scopus Google Scholar). We many proteins that over the cell cycle their post-translational through the The between transcriptional regulation and regulation protein is the for the cell to a is the in and of the is regulation an and to control protein We the phosphorylation events over the cell cycle, and a of these to protein and transcript dynamics. the post-translational regulation of protein activity, the localization a and the of the cell as a to separate protein functions Our to the and proteomic cell cycle for the cell cycle We for proteins in this a in of a larger a in to a larger or a between with different The can for the of and as are to the of the in and the the unique of proteins that as in the cell cycle a of proteins, the of that during M. M. the cell division Biol. 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