A quantitative atlas of mitotic phosphorylation

TLDR

The eukaryotic cell division cycle is a highly regulated sequence of events that duplicates and separates cellular material, driven by protein phosphorylation mediated by cyclin‑dependent kinases. This study aimed to identify proteins whose phosphorylation is regulated across the cell cycle. The authors employed stable isotope labeling, a two‑step phosphopeptide enrichment protocol, and high‑accuracy mass spectrometry to analyze phosphorylation in a human cell line arrested in G1 and mitosis. They quantified over 14,000 phosphorylation events—more than half novel—found that >1,000 proteins are hyperphosphorylated in mitosis, most sites are CDK‑consensus SP motifs, and two distinct non‑proline motifs suggest the existence of at least two previously uncharacterized mitotic kinases.

Abstract

The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G(1) and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.