Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/636407

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

Yamazaki, H.·Kosako, H.·Yoshimura, S. H.
View original source ↗Export metadata ↓

Abstract

Protein phosphorylation plays a critical role in the regulation and progression of mitosis. More than 10,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear. In this study, we examined structural properties of mitotic phosphosites and neighboring residues to understand the role of heavy phosphorylation in non-structured domains. Quantitative mass spectrometry analysis of mitosis-arrested and non-arrested HeLa cells revealed >4,100 and >2,200 residues either significantly phosphorylated or dephosphorylated, respectively, at mitotic entry. The calculated disorder scores of amino acid sequences of neighboring individual phosphosites revealed that >70% of dephosphorylated phosphosites exist in disordered regions, whereas 50% of phosphorylated sites exist in non-structured domains. A clear inverse correlation was observed between probability of phosphorylation in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at entry to mitosis, a significant number of phosphate groups are removed from non-structured domains and transferred to more-structured domains. Gene ontology term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, suggesting that heavy phosphorylation/dephosphorylation in non-structured domains of RNA-binding proteins plays a role in dynamic rearrangement of RNA-containing organelles, as well as other intracellular environments.\n\nSignificance StatementProgression of mitosis is tightly regulated by protein phosphorylation/dephosphorylation. Although proteomic studies have identified tens of thousands of phosphosites in mitotic cells, the roles of them remain to be answered. We approached this question from the viewpoint of the higher-order structure of phosphosites. Quantitative proteomics and bioinformatic analyses revealed that more than 70% of mitotic dephosphorylation events occurred in non-structured regions. Non-structured regions of cellular proteins are attracting considerable attention in terms of their involvement in dynamic rearrangements of intracellular membrane-less organelles and protein assembly/disassembly processes. Our results suggest the possibility that a vast amount of mitosis-associated dephosphorylation/phosphorylation at non-structured regions plays a role in regulating the dynamic assembly/disassembly of intracellular architectures and organelles such as chromosomes and nucleolus.

Source connections

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Yamazaki, H., Kosako, H., Yoshimura, S. H.. 2019-05-13. Quantitative proteomics revealed a tight correlation between mitotic phosphorylation/dephosphorylation and structural properties of substrate domains. https://doi.org/10.1101/636407

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Endothelial Cell Specific Thioredoxin 1-Dependent Signaling in Oxygen-1 Induced Retinopathy

Purpose: Retinopathy of prematurity (ROP) is a leading cause of childhood blindness, characterized byoxidative stress and dysregulated angiogenesis in preterm infants with immature antioxidant systems.This study aimed to investigate the specific role of endothelial cell-specific thioredoxin 1 (TXN1), a crucial oxidoreductase, in oxygen-induced retinopathy, hypothesizing that the absence of endothelial TXN1 directly impairs angiogenesis and exacerbates hyperoxia-induced injury. Methods: We generated tamoxifen-inducible, endothelial cell-specific Txn1 knockout mice (Txn1-iECKO) and utilized the oxygen-induced retinopathy (OIR) model, exposing litters to 85% oxygen from postnatal day 7 (P7) to P12. Retinal vascular morphology during vaso-obliteration and neovascularization phases, endothelial cell proliferation, and apoptotic cell death were assessed. Furthermore, human retinal microvascular endothelial cells (HRMECs) with TXN1 knockdown were subjected to hyperoxic conditions, followed by comprehensive gene expression profiling using RT2 Profiler PCR arrays and Ingenuity Pathway Analysis. Results: Deletion of endothelial Txn1 significantly enhanced vascular regression during the hyperoxic vaso-obliteration phase at P12 and markedly exacerbated pathological neovascularization at P17. Txn1-deficient retinas exhibited significantly impaired endothelial cell proliferation during the vaso-obliteration phase, while apoptotic cell death remained unchanged. Transcriptomic analysis of TXN1-deficient HRMECs under hyperoxia revealed an enhanced activation of NRF2-mediated oxidative stress response pathways. Conclusions: These findings identify endothelial TXN1 as an essential and oxygen-dependent regulator of vascular development in ischemic retinopathy, primarily influencing endothelial proliferation and redox signaling. This suggests TXN1's crucial protective role and highlights its therapeutic potential to mitigate ROP progression by modulating endothelial cell responses to oxidative stress.

cell biology↗
bioRxiv2026

A Dynamic Stem Cell State Links Environmental Cues to Skeletal and Regenerative Fates

Stem cells faithfully generate tissue architecture during development, yet how their identity and regenerative output are reshaped as anatomical niches mature and environmental signals change with aging and injury remains poorly understood. Rare skeletal stem cells (SSCs) across diverse skeletal sites are defined by combinations of cell-surface markers, precluding their genetic interrogation in vivo. Here, using spatially informed single cell transcriptomics, we identify Pannexin-3 (Panx3) to mark a dynamic functional state within bona fide SSCs and establish Panx3-CreERT2 mice for its longitudinal interrogation. Panx3 SSCs exhibit long-term clonal persistence, self-renewal and multipotency, whereas Panx3- SSCs can acquire this state. Successive SSC cohorts engage the Panx3 state during skeletal development, osteoanabolic stimulation expands pre-existing Panx3 SSC clones and injury induces it locally in previously unlabeled progenitors. Thus, anatomically distinct progenitors can converge on a common stem cell state in response to different environmental inputs. Three-dimensional reconstruction of chondrogenic niches further identifies signals that redirect Panx3-lineage cells towards stable cartilage, enabling durable regeneration in aged and osteoarthritic joints and with human SSCs. These findings establish anatomical origin, stem cell state and lineage fate as separable dimensions of stem cell identity and provide genetic access for interrogating and directing endogenous SSC function.

cell biology↗
bioRxiv2026

Proteome-wide profiling reveals compartment-specific RNA buffering of protein aggregation during stress

Cellular adaptation to stress requires extensive remodeling of RNA metabolism. The rewiring of the RNA-protein interaction landscape and the assembly of biomolecular condensates are central to this response, yet the global stress-induced reorganization of macromolecular RNA-protein assemblies remains incompletely characterized. Here, we apply R-DeeP, a density gradient approach coupled to quantitative mass spectrometry, to profile the RNA-dependent sedimentation behaviour of the Saccha-romyces cerevisiae proteome during heat and salt stress. We demonstrate that stress triggers a profound, stress-specific reorganization of the RNA interactome. Many proteins undergo pronounced, selective shifts into high-molecular weight fractions, indicative of aggregation. RNase treatment markedly exacerbates this aggregation, consistent with the model that RNA acts as a general chaperone that keeps proteins with intrinsically disordered regions soluble. Strikingly, our data demonstrate that this protection is compartmentalised. Nuclear proteins are partially shielded from stress-induced aggregation. This protection coincides with the nuclear accumulation of RNA caused by a block of RNA export during stress, and RNase treatment abolishes this protection. Nuclear RNA retention may therefore act as a buffer that actively increases the local chaperoning capacity of the nucleus. In contrast, cytoplasmic proteins, including translation factors and ribonucleoprotein granule components, exhibit increased aggregation under stress. Fluorescence microscopy confirmed that increased biochemical aggregation in density gradients reflects changes in assembly state in vivo. For many stress-induced aggregators, these changes are visible as spatial reorganisation in stressed cells, such as the formation of foci. Our findings suggest that cells use subcellular RNA distribution to tune the condensation and/or aggregation threshold of proteins in a compartment-specific manner.

cell biology↗