Quantitative proteomics revealed a tight correlation between mitotic phosphorylation/dephosphorylation and structural properties of substrate domains

Abstract Protein phosphorylation plays inevitable roles in the regulation and the progression of mitosis. More than ten thousands of phosphorylated residues and their responsible kinases have so far been identified by a number of proteomic analyses. While some of those phosphosites have been demonstrated to affect either protein-protein interaction or catalytic activity of the substrate protein, the effect of most mitotic phosphosites remain unclarified. In this study, we tried to extract structural properties of mitotic phosphosites and neighboring residues, and find a role of heavy phosphorylation at non-structured domain. A quantitative mass spectrometry of mitosis-arrested and non-arrested HeLa cells identified more than 4,100 and 1,900 residues which are significantly phosphorylated, and dephosphorylated at mitotic entry, respectively. Calculating the disorder score of the neighboring amino acid sequence of individual phosphosites revealed that more than 70% of the phosphosites which are dephosphorylated existed in disordered regions, whereas 50% of phosphorylated sites existed in non-structured domain. There was a clear inversed co-relation between probability in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at the entry of mitosis, a significant amount of phosphate group is removed from non-structured domains and attached to more structured domains. GO term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, implying that heavy phosphorylation/dephosphorylation in non-structured domain of RNA-binding proteins may play a role in dynamic rearrangement of RNA-containing organelle, as well as other intracellular environments.