Dataset Information

Identification of mitotic substrates of phospho-regulated USP9X

ABSTRACT: We previously identified the deubiquitylase USP9X as a G2/M-specific CDC14B interactor (PXD012732) and subsequently identified USP9X as a specific CDC14B target (PXD012733). To investigate relevant mitotic substrates of phospho-regulated USP9X, we next performed a Stable isotope labeling by amino acids in cell culture (SILAC)-based screen in which ubiquitylated proteins were purified from control or USP9X depleted HEK 293T cells that were either asynchronous or synchronized in mitosis. Stable expression of recombinant His-tagged ubiquitin has been used for selective enrichment of ubiquitinylated proteins in both conditions.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Johannes Gloeckner

LAB HEAD: Christian Johannes Gloeckner

PROVIDER: PXD012734 | Pride | 2020-03-25

REPOSITORIES: Pride

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Publications

Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival.

Dietachmayr Michael M Rathakrishnan Abirami A Karpiuk Oleksandra O von Zweydorf Felix F Engleitner Thomas T Fernández-Sáiz Vanesa V Schenk Petra P Ueffing Marius M Rad Roland R Eilers Martin M Gloeckner Christian Johannes CJ Clemm von Hohenberg Katharina K Bassermann Florian F

Nature communications 20200309 1

Regulation of mitosis secures cellular integrity and its failure critically contributes to the development, maintenance, and treatment resistance of cancer. In yeast, the dual phosphatase Cdc14 controls mitotic progression by antagonizing Cdk1-mediated protein phosphorylation. By contrast, specific mitotic functions of the mammalian Cdc14 orthologue CDC14B have remained largely elusive. Here, we find that CDC14B antagonizes CDK1-mediated activating mitotic phosphorylation of the deubiquitinase U ...[more]

PMID: 32152317

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Project description:Post translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Since little is known about crosstalk between different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk between different PTMs. In addition various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin upon inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, while the total protein level decreased immediately afterwards. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.

Project description:Ubiquitination has crucial roles in many cellular processes and dysregulation of ubiquitin machinery enzymes can result in various forms of pathogenesis. Cells only have a limited set of E2 ubiquitin-conjugating enzymes to support the ubiquitination of many cellular targets. As individual E2 enzymes have many different substrates and interactions between E2 enzymes and their substrates can be transient, it is challenging to define all in vivo substrates of an individual E2 and the cellular processes it affects. Particularly challenging in this respect is UBE2D3, a very promiscuous E2 enzyme that affects a broad range of processes. Here, we set out to identify in vivo targets of UBE2D3 by using SILAC-based quantitative ubiquitin diGly proteomics to study global proteome and ubiquitinome changes associated with UBE2D3 depletion. UBE2D3 depletion changed the global proteome, with proteins from metabolic pathways, in particular retinol metabolism, being the most affected. However, the impact of UBE2D3 depletion on the ubiquitinome was much more prominent and, interestingly, identified pathways related to mRNA translation as top pathways being affected. Indeed, we find that ubiquitination of the ribosomal proteins RPS10 and RPS20, critical for ribosome-associated protein quality control (RQC), is dependent on UBE2D3. Moreover, we find that UBE2D3 limits monoubiquitination of histone H2B at lysine 120, mediated by the E3 ligase RNF20. This identifies UBE2D3 as a potential regulator of the diverse and critical cellular processes that are controlled by H2B monoubiquitination. Collectively, our findings show that depletion of an E2 ubiquitin-conjugating enzyme in combination with quantitative diGly-based ubiquitinome profiling is a powerful tool to identify new in vivo E2 substrates, as we have done here for UBE2D3. Together with our identification of RQC factors and H2B as novel in vivo targets of UBE2D3, our work provides an important resource for further studies on the in vivo functions of UBE2D3.

Dataset Information

Identification of mitotic substrates of phospho-regulated USP9X

Publications

Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival.

Similar Datasets

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