Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

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.