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Updated: Jun 17, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis
Jesper V Olsen1, Michiel Vermeulen, Anna Santamaria
1Department of Proteomics and Signal Transduction, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried near Munich, Germany.
Abstract:
Eukaryotic cells replicate by a complex series of evolutionarily conserved events that are tightly regulated at defined stages of the cell division cycle. Progression through this cycle involves a large number of dedicated protein complexes and signaling pathways, and deregulation of this process is implicated in tumorigenesis. We applied high-resolution mass spectrometry-based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics. Co-regulated proteins and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global activation during S phase of the DNA damage response network, which was mediated by phosphorylation by ATM or ATR or DNA-dependent protein kinases. We determined site-specific stoichiometry of more than 5000 sites and found that most of the up-regulated sites phosphorylated by cyclin-dependent kinase 1 (CDK1) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells.
Insights
This study mapped the human cell cycle proteome and phosphoproteome, revealing significant protein and phosphorylation dynamics, especially during mitosis. Key findings highlight the cell cycle regulation of DNA damage response and protein inactivation via phosphorylation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Eukaryotic cell replication is a highly regulated process crucial for cell division.
- Dysregulation of the cell cycle is linked to cancer development.
- Understanding cell cycle proteome and phosphoproteome dynamics is essential for comprehending cellular function and disease.
Purpose of the Study:
- To globally investigate the human cell cycle proteome and phosphoproteome using high-resolution mass spectrometry.
- To quantify protein and phosphorylation site dynamics throughout the cell cycle.
- To compare proteomic data with messenger RNA expression data.
Main Methods:
- High-resolution mass spectrometry-based proteomics and phosphoproteomics.
- Global quantification of proteins and phosphorylation sites.
- Analysis of cell cycle kinetics and comparison with mRNA microarray data.
Main Results:
- Quantified 6027 proteins and 20,443 unique phosphorylation sites, revealing substantial regulation, particularly in mitotic cells.
- Identified global activation of the DNA damage response network during S phase, mediated by specific kinases.
- Determined site-specific stoichiometry, showing high phosphorylation occupancy for cyclin-dependent kinase 1 (CDK1) and CDK2 targets in mitosis, potentially inactivating nuclear and metabolic proteins.
Conclusions:
- The human cell cycle proteome and phosphoproteome exhibit significant dynamic regulation, especially during mitosis.
- Phosphorylation plays a critical role in cell cycle control, including DNA damage response and regulation of protein activity.
- Specific proteins, including nuclear and metabolic regulators, appear to be inactivated by phosphorylation during mitosis.
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