Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates

Abstract

16Protein phosphorylation plays inevitable roles in the regulation and the progression of mitosis. More than ten 17 thousands of phosphorylated residues and their responsible kinases have so far been identified by a number of 18 proteomic analyses. While some of those phosphosites have been demonstrated to affect either protein-protein 19 interaction or catalytic activity of the substrate protein, the effect of most mitotic phosphosites remain 20 unclarified. In this study, we tried to extract structural properties of mitotic phosphosites and neighboring 21 residues, and find a role of heavy phosphorylation at non-structured domain. A quantitative mass spectrometry 22 of mitosis-arrested and non-arrested HeLa cells identified more than 4,100 and 1,900 residues which are 23 significantly phosphorylated, and dephosphorylated at mitotic entry, respectively. Calculating the disorder score 24 of the neighboring amino acid sequence of individual phosphosites revealed that more than 70% of the 25 phosphosites which are dephosphorylated existed in disordered regions, whereas 50% of phosphorylated sites 26 existed in non-structured domain. There was a clear inversed co-relation between probability in non-structured 27 domain and increment of phosphorylation in mitosis. These results indicate that at the entry of mitosis, a 28 significant amount of phosphate group is removed from non-structured domains and attached to more structured 29 domains. GO term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-30 related proteins are both dephosphorylated and phosphorylated, implying that heavy 31 phosphorylation/dephosphorylation in non-structured domain of RNA-binding proteins may play a role in 32 dynamic rearrangement of RNA-containing organelle, as well as other intracellular environments. 33 34 65been identified (reviewed in (17-21)). Dephosphorylation also plays essential roles both at the entry and the 66 exit of mitosis (22, 23) (24, 25). More than 1,000 kinds of phosphosites are dephosphorylated during anaphase 67 (26), and more than five hundreds are dephosphorylated at the entry of mitosis (2). For example, CDK1 is 68 activated by dephosphorylation by Cdc25B/C (27, 28), and Cdc25C is partially activated by protein phosphatase 69 1 (27). These results indicate that a vast number of phosphate groups are transferred to and from the substrate 70 proteins during mitotic entry. 71One of the unclarified problems on mitotic phosphorylation could be why and how a vast number of 72 phosphate groups are transferred from a set of proteins to a different set of proteins, and whether these different 73 sets of mitotic phosphosites are structurally different or not. Especially, the distribution of phosphosites in 74 structured or non-structured regions of the substrate proteins is an important cue to understand the structural 75 effect of phosphorylation/dephosphorylation. In this study, we performed phosphoproteomic analysis of cellular 76 proteins to quantify and compare individual phosphosi...

How Do Flaviviruses Hijack Host Cell Functions by Phase Separation?

“…The NS5 of flaviviruses is a conserved-phospho-protein [ 108 , 109 , 110 , 111 ] that is reportedly phosphorylated by casein kinase I [ 112 ] or protein kinase G [ 113 ]. In general, phosphorylation predominantly occurs to the IDRs [ 114 , 115 , 116 ], which regulates phase separation and intracellular RNA granule dynamics [ 117 , 118 , 119 , 120 ]. Although the detailed mechanism of phosphorylation-dependent regulation of phase separation has not yet been clarified, the segregation of charged residues along the polypeptide chain (linear distribution of oppositely charged blocks) plays a critical role in enhancing phase separation [ 121 , 122 , 123 , 124 ].…”