Quantitative Phosphoproteomics Reveals Widespread Full Phosphorylation Site Occupancy During Mitosis

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.

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

“…18,76 Much greater effects on phosphorylation throughout the entire signaling cascade are also observed, as expected. In the Rap signaling pathway, we observe that the majority of phosphorylation events occur much further downstream in structural components of the cell such as adherens junction proteins or actin cytoskeleton-associated proteins.…”

Mitotic phosphorylation of CCCTC‐binding factor (CTCF) reduces its DNA binding activity

“…Supernatant and pellet fractions were obtained by centrifugation at 2300 g for 5 min. [9] and [10]. Blue and green characters indicate phosphorylation sites analyzed in [11] and [17], respectively.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…Some of these sites may be more suitable markers of catalytic activation. identify proteins whose phosphorylation state is regulated by the cell cycle [102][103][104] and (c) indentify proteins whose phosphorylation state is perturbed as a consequence of drug/inhibitor treatment [105,106]. As phosphoproteomics studies and indeed smaller, conventional studies continue to identify new phosphorylation sites on PKC…”