HAT HBO1 interacts with 2 isoforms of JADE1: JADE1S and JADE1L. JADE1 promotes acetylation of nucleosomal histones by HBO1. HBO1–JADE1 complex facilitates cell proliferation by unclear mechanisms. Here we report intracellular chromatin shuttling of HBO1–JADE1 complex during mitosis coupled to phosphorylation of JADE1. In interphase of dividing cells JADE1S was localized to the nucleus and associated with chromatin. As cells approached mitosis, specifically prophase, JADE1S dissociated from chromatin and associated with cytoplasm. JADE1S chromatin re-association began in telophase and paralleled nuclear envelope membrane reassembly. By early G1, JADE1S was re-associated with chromatin and localized to the nucleus. Importantly, cytoplasmic but not chromatin-associated JADE1 protein was phosphorylated. Mass-Spectrometric analysis of JADE1S protein isolated from G2/M-arrested cells identified 6 phosphorylated amino acid residues: S89, T92, S102, S121, S392, and T468, including 3 novel sites. Temporally, JADE1S phosphorylation and dephosphorylation during mitosis correlated with JADE1S chromatin dissociation and recruitment. JADE1S chromatin recruitment was accompanied by the global histone H4 acetylation. Pharmacological inhibitor of Aurora A kinase prevented JADE1S protein band shift and chromatin dissociation, suggesting regulatory function for phosphorylation. In vivo experiments supported our in vitro results. In mouse kidneys, JADE1S transiently accumulated in the cytoplasm of tubular epithelial cells during kidney regeneration. The transient increase in the number of cells with cytoplasmic JADE1S directly correlated with activation of tubular cell proliferation and inversely correlated with the number of cells with nuclear JADE1S staining, supporting biological role of HBO1–JADE1 shuttling during organ regeneration.
JADE1 belongs to a small family of PHD zinc finger proteins that interacts with histone acetyl transferase (HAT) HBO1 and is associated with chromatin. We recently reported JADE1 chromatin shuttling and phosphorylation during G2/M to G1 transition, which was sensitive to Aurora A inhibition. In the current study we examined mechanisms of the cell cycle regulation by the small isoform of JADE1 protein, JADE1S, and report data showing that JADE1S has a novel function in the regulation of cytokinesis. Using FACS assays, we show that, JADE1S depletion facilitated rates of G1-cells accumulation in synchronously dividing HeLa cell cultures. Depletion of JADE1S protein in asynchronously dividing cells decreased the proportion of cytokinetic cells, and increased the proportion of multi-nuclear cells, indicative of premature and failed cytokinesis. In contrast, moderate overexpression of JADE1S increased the number of cytokinetic cells in time-and dose-dependent manner, indicating cytokinetic delay. Pharmacological inhibition of Aurora B kinase resulted in the release of JADE1S-mediated cytokinetic delay and allowed progression of abscission in cells overexpressing JADE1S. Finally, we show that JADE1S protein localized to centrosomes in interphase and mitotic cells, while during cytokinesis JADE1S localized to the midbody. Neither JADE1L nor partner of JADE1, HAT HBO1 was localized to the centrosomes or midbodies. Our study identifies the novel role for JADE1S in regulation of cytokinesis and suggests function in Aurora B kinase-mediated cytokinesis checkpoint.
Determination of target engagement for candidate drug molecules in the native cellular environment is a significant challenge for drug discovery programs. The cellular thermal shift assay (CETSA) has emerged as a powerful tool for determining compound target engagement through measurement of changes to a protein's thermal stability upon ligand binding. Here, we present a HiBiT thermal shift assay (BiTSA) that deploys a quantitative peptide tag for determination of compound target engagement in the native cellular environment using a high throughput, plate-based luminescence readout. We demonstrate that BiTSA can rapidly assess cellular target engagement of small molecule ligands against their cognate targets and highlight two applications of BiTSA for differentiating small molecules targeting mutant KRAS and TP53.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.