Publications by authors named "Akinori Endo"

17 Publications

  • Page 1 of 1

TRIP12 promotes small-molecule-induced degradation through K29/K48-branched ubiquitin chains.

Mol Cell 2021 04 9;81(7):1411-1424.e7. Epub 2021 Feb 9.

Institute for Advanced Life Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan; School of Pharmacy and Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan; Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. Electronic address:

Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2 substrate HIF-1α. TRIP12 associates with BRD4 via CRL2 and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.
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http://dx.doi.org/10.1016/j.molcel.2021.01.023DOI Listing
April 2021

Multi-Step Ubiquitin Decoding Mechanism for Proteasomal Degradation.

Pharmaceuticals (Basel) 2020 Jun 23;13(6). Epub 2020 Jun 23.

Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.

The 26S proteasome is a 2.5-MDa protease complex responsible for the selective and ATP-dependent degradation of ubiquitylated proteins in eukaryotic cells. Proteasome-mediated protein degradation accounts for ~70% of all cellular proteolysis under basal conditions, and thereby any dysfunction can lead to drastic changes in cell homeostasis. A major function of ubiquitylation is to target proteins for proteasomal degradation. Accompanied by deciphering the structural diversity of ubiquitin chains with eight linkages and chain lengths, the ubiquitin code for proteasomal degradation has been expanding beyond the best-characterized Lys48-linked ubiquitin chains. Whereas polyubiquitylated proteins can be directly recognized by the proteasome, in several cases, these proteins need to be extracted or segregated by the conserved ATPases associated with diverse cellular activities (AAA)-family ATPase p97/valosin-containing protein (VCP) complex and escorted to the proteasome by ubiquitin-like (UBL)-ubiquitin associated (UBA) proteins; these are called substrate-shuttling factors. Furthermore, proteasomes are highly mobile and are appropriately spatiotemporally regulated in response to different cellular environments and stresses. In this review, we highlight an emerging key link between p97, shuttling factors, and proteasome for efficient proteasomal degradation. We also present evidence that proteasome-containing nuclear foci form by liquid-liquid phase separation under acute hyperosmotic stress.
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http://dx.doi.org/10.3390/ph13060128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344625PMC
June 2020

RQT complex dissociates ribosomes collided on endogenous RQC substrate SDD1.

Nat Struct Mol Biol 2020 04 23;27(4):323-332. Epub 2020 Mar 23.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.
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http://dx.doi.org/10.1038/s41594-020-0393-9DOI Listing
April 2020

Nik-related kinase is targeted for proteasomal degradation by the chaperone-dependent ubiquitin ligase CHIP.

FEBS Lett 2020 06 1;594(11):1778-1786. Epub 2020 Apr 1.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.

Nik-related kinase (Nrk) is a member of the germinal center kinase IV family and suppresses Akt signaling. In vivo, Nrk prevents placental hyperplasia and breast cancer formation. Here, we show that Nrk is regulated by the chaperone-dependent ubiquitin ligase carboxyl terminus of heat-shock protein (Hsp)70-interacting protein (CHIP). Immunoprecipitation and liquid chromatography-tandem mass spectrometry analysis reveal that Nrk preferentially interacts with CHIP and Hsp70/90 family proteins. Nrk protein levels are decreased by CHIP overexpression and increased by siRNA-mediated CHIP knockdown. Our results indicate that Nrk is ubiquitinated by CHIP in a chaperone-dependent manner, resulting in its proteasomal degradation. CHIP targets a fraction of Nrk molecules that have lost the ability to regulate Akt signaling. We conclude that CHIP plays an important role in regulating Nrk protein levels.
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http://dx.doi.org/10.1002/1873-3468.13769DOI Listing
June 2020

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Nat Commun 2020 03 6;11(1):1222. Epub 2020 Mar 6.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
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http://dx.doi.org/10.1038/s41467-020-15006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060239PMC
March 2020

Stress- and ubiquitylation-dependent phase separation of the proteasome.

Nature 2020 02 5;578(7794):296-300. Epub 2020 Feb 5.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.

The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.
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http://dx.doi.org/10.1038/s41586-020-1982-9DOI Listing
February 2020

The integral function of the endocytic recycling compartment is regulated by RFFL-mediated ubiquitylation of Rab11 effectors.

J Cell Sci 2019 02 7;132(3). Epub 2019 Feb 7.

Department of Biomedical Chemistry, School of Science and Technology, Kwansei Gakuin University, Hyogo 669-1337, Japan

Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors , but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.
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http://dx.doi.org/10.1242/jcs.228007DOI Listing
February 2019

Ubiquitin-specific protease 8 deubiquitinates Sec31A and decreases large COPII carriers and collagen IV secretion.

Biochem Biophys Res Commun 2018 05 31;499(3):635-641. Epub 2018 Mar 31.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan; Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, 226-8501, Japan. Electronic address:

Nascent cargo proteins in the endoplasmic reticulum are transported to the Golgi by COPII carriers. Typical COPII vesicles are 60-70 nm in diameter, and much larger macromolecules, such as procollagen, are transported by atypical large COPII carriers in mammalian cells. The formation of large COPII carriers is enhanced by Cul3 ubiquitin ligase, which mono-ubiquitinates Sec31A, a COPII coat protein. However, the deubiquitinating enzyme for Sec31A was unclear. Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A. The interaction was mediated by the adaptor protein STAM1. USP8 overexpression inhibited the formation of large COPII carriers. By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion. We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion.
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http://dx.doi.org/10.1016/j.bbrc.2018.03.202DOI Listing
May 2018

Deubiquitylases USP5 and USP13 are recruited to and regulate heat-induced stress granules through their deubiquitylating activities.

J Cell Sci 2018 04 12;131(8). Epub 2018 Apr 12.

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8501, Japan

Stress granules are transient cytoplasmic foci induced by various stresses that contain translation-stalled mRNAs and RNA-binding proteins. They are proposed to modulate mRNA translation and stress responses. Here, we show that the deubiquitylases USP5 and USP13 are recruited to heat-induced stress granules. Heat-induced stress granules also contained K48- and K63-linked ubiquitin chains. Depletion of USP5 or USP13 resulted in elevated ubiquitin chain levels and accelerated assembly of heat-induced stress granules, suggesting that these enzymes regulate the stability of the stress granules through their ubiquitin isopeptidase activity. Moreover, disassembly of heat-induced stress granules after returning the cells to normal temperatures was markedly repressed by individual depletion of USP5 or USP13. Finally, overexpression of a ubiquitin mutant lacking the C-terminal diglycine motif caused the accumulation of unanchored ubiquitin chains and the repression of the disassembly of heat-induced stress granules. As unanchored ubiquitin chains are preferred substrates for USP5, we suggest that USP5 regulates the assembly and disassembly of heat-induced stress granules by mediating the hydrolysis of unanchored ubiquitin chains while USP13 regulates stress granules through deubiquitylating protein-conjugated ubiquitin chains.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.210856DOI Listing
April 2018

Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation.

EMBO Rep 2017 03 11;18(3):403-419. Epub 2017 Jan 11.

Institute of Medical Sciences, School of Medicine, Medical Sciences & Nutrition, University of Aberdeen, Aberdeen, UK

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation-mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N-terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1-PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1-PP1 protects the origin-binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1-depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1-targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.
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http://dx.doi.org/10.15252/embr.201641983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331243PMC
March 2017

The Chromatin Assembly Factor Complex 1 (CAF1) and 5-Azacytidine (5-AzaC) Affect Cell Motility in Src-transformed Human Epithelial Cells.

J Biol Chem 2017 Jan 21;292(1):172-184. Epub 2016 Nov 21.

From the Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom

Tumor invasion into surrounding stromal tissue is a hallmark of high grade, metastatic cancers. Oncogenic transformation of human epithelial cells in culture can be triggered by activation of v-Src kinase, resulting in increased cell motility, invasiveness, and tumorigenicity and provides a valuable model for studying how changes in gene expression cause cancer phenotypes. Here, we show that epithelial cells transformed by activated Src show increased levels of DNA methylation and that the methylation inhibitor 5-azacytidine (5-AzaC) potently blocks the increased cell motility and invasiveness induced by Src activation. A proteomic screen for chromatin regulators acting downstream of activated Src identified the replication-dependent histone chaperone CAF1 as an important factor for Src-mediated increased cell motility and invasion. We show that Src causes a 5-AzaC-sensitive decrease in both mRNA and protein levels of the p150 (CHAF1A) and p60 (CHAF1B), subunits of CAF1. Depletion of CAF1 in untransformed epithelial cells using siRNA was sufficient to recapitulate the increased motility and invasive phenotypes characteristic of transformed cells without activation of Src. Maintaining high levels of CAF1 by exogenous expression suppressed the increased cell motility and invasiveness phenotypes when Src was activated. These data identify a critical role of CAF1 in the dysregulation of cell invasion and motility phenotypes seen in transformed cells and also highlight an important role for epigenetic remodeling through DNA methylation for Src-mediated induction of cancer phenotypes.
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http://dx.doi.org/10.1074/jbc.M116.751024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217677PMC
January 2017

MYC-dependent recruitment of RUNX1 and GATA2 on the SET oncogene promoter enhances PP2A inactivation in acute myeloid leukemia.

Oncotarget 2017 Aug 6;8(33):53989-54003. Epub 2016 Jun 6.

Hematology/Oncology Program, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain.

The SET (I2PP2A) oncoprotein is a potent inhibitor of protein phosphatase 2A (PP2A) that regulates many cell processes and important signaling pathways. Despite the importance of SET overexpression and its prognostic impact in both hematologic and solid tumors, little is known about the mechanisms involved in its transcriptional regulation. In this report, we define the minimal promoter region of the gene, and identify a novel multi-protein transcription complex, composed of MYC, SP1, RUNX1 and GATA2, which activates expression in AML. The role of MYC is crucial, since it increases the expression of the other three transcription factors of the complex, and supports their recruitment to the promoter of . These data shed light on a new regulatory mechanism in cancer, in addition to the already known PP2A-MYC and SET-PP2A. Besides, we show that there is a significant positive correlation between the expression of and , , and in AML patients, which further endorses our results. Altogether, this study opens new directions for understanding the mechanisms that lead to overexpression, and demonstrates that MYC, SP1, RUNX1 and GATA2 are key transcriptional regulators of expression in AML.
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http://dx.doi.org/10.18632/oncotarget.9840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589557PMC
August 2017

Analysis of human protein replacement stable cell lines established using snoMEN-PR vector.

PLoS One 2013 25;8(4):e62305. Epub 2013 Apr 25.

Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, United Kingdom.

The study of the function of many human proteins is often hampered by technical limitations, such as cytotoxicity and phenotypes that result from overexpression of the protein of interest together with the endogenous version. Here we present the snoMEN (snoRNA Modulator of gene ExpressioN) vector technology for generating stable cell lines where expression of the endogenous protein can be reduced and replaced by an exogenous protein, such as a fluorescent protein (FP)-tagged version. SnoMEN are snoRNAs engineered to contain complementary sequences that can promote knock-down of targeted RNAs. We have established and characterised two such partial protein replacement human cell lines (snoMEN-PR). Quantitative mass spectrometry was used to analyse the specificity of knock-down and replacement at the protein level and also showed an increased pull-down efficiency of protein complexes containing exogenous, tagged proteins in the protein replacement cell lines, as compared with conventional co-expression strategies. The snoMEN approach facilitates the study of mammalian proteins, particularly those that have so far been difficult to investigate by exogenous expression and has wide applications in basic and applied gene-expression research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062305PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636044PMC
November 2013

Human box C/D snoRNA processing conservation across multiple cell types.

Nucleic Acids Res 2012 Apr 22;40(8):3676-88. Epub 2011 Dec 22.

Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

Small nucleolar RNAs (snoRNAs) function mainly as guides for the post-transcriptional modification of ribosomal RNAs (rRNAs). In recent years, several studies have identified a wealth of small fragments (<35 nt) derived from snoRNAs (termed sdRNAs) that stably accumulate in the cell, some of which may regulate splicing or translation. A comparison of human small RNA deep sequencing data sets reveals that box C/D sdRNA accumulation patterns are conserved across multiple cell types although the ratio of the abundance of different sdRNAs from a given snoRNA varies. sdRNA profiles of many snoRNAs are specific and resemble the cleavage profiles of miRNAs. Many do not show characteristics of general RNA degradation, as seen for the accumulation of small fragments derived from snRNA or rRNA. While 53% of the sdRNAs contain an snoRNA box C motif and boxes D and D' are also common in sdRNAs (54%), relatively few (12%) contain a full snoRNA guide region. One box C/D snoRNA, HBII-180C, was analysed in greater detail, revealing the presence of C' box-containing sdRNAs complementary to several pre-messenger RNAs (pre-mRNAs) including FGFR3. Functional analyses demonstrated that this region of HBII-180C can influence the alternative splicing of FGFR3 pre-mRNA, supporting a role for some snoRNAs in the regulation of splicing.
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http://dx.doi.org/10.1093/nar/gkr1233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3333852PMC
April 2012

[Cellular functions of the deubiquitylating enzyme family].

Seikagaku 2010 May;82(5):378-87

Department of Biological Sciences, Tokyo Institute of Technology, 4259-B16 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.

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May 2010

Nucleophosmin/B23 regulates ubiquitin dynamics in nucleoli by recruiting deubiquitylating enzyme USP36.

J Biol Chem 2009 Oct 13;284(41):27918-27923. Epub 2009 Aug 13.

Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226-8501, Japan. Electronic address:

The nucleolus is a subnuclear compartment with multiple cellular functions, including ribosome biogenesis. USP36 is a deubiquitylating enzyme that localizes to nucleoli and plays an essential role in regulating the structure and function of the organelle. However, how the localization of USP36 is regulated remains unknown. Here, we identified a short stretch of basic amino acids (RGKEKKIKKFKREKRR) that resides in the C-terminal region of USP36 and serves as a nucleolar localization signal for the protein. We found that this motif interacts with a central acidic region of nucleophosmin/B23, a major nucleolar protein involved in various nucleolar functions. Knockdown of nucleophosmin/B23 resulted in a significant reduction in the amount of USP36 in nucleoli, without affecting the cellular USP36 level. This was associated with elevated ubiquitylation levels of fibrillarin, a USP36 substrate protein in nucleoli. We conclude that nucleophosmin/B23 recruits USP36 to nucleoli, thereby serving as a platform for the regulation of nucleolar protein functions through ubiquitylation/deubiquitylation.
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http://dx.doi.org/10.1074/jbc.M109.037218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788843PMC
October 2009

Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36.

J Cell Sci 2009 Mar 10;122(Pt 5):678-86. Epub 2009 Feb 10.

Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

The nucleolus is a subnuclear compartment and the site of ribosome biogenesis. Previous studies have implicated protein ubiquitylation in nucleolar activity. Here we show that USP36, a deubiquitylating enzyme of unknown function, regulates nucleolar activity in mammalian cells. USP36 localized to nucleoli via the C-terminal region, which contains basic amino acid stretches. Dominant-negative inhibition of USP36 caused the accumulation of ubiquitin-protein conjugates in nucleoli, suggesting that nucleoli are the site of USP36 action. USP36 deubiquitylated the nucleolar proteins nucleophosmin/B23 and fibrillarin, and stabilized them by counteracting ubiquitylation-mediated proteasomal degradation. RNAi-mediated depletion of cellular USP36 resulted in reduced levels of rRNA transcription and processing, a less-developed nucleolar morphology and a slight reduction in the cytoplasmic ribosome level, which eventually led to a reduced rate of cell proliferation. We conclude that by deubiquitylating various nucleolar substrate proteins including nucleophosmin/B23 and fibrillarin, USP36 plays a crucial role in regulating the structure and function of nucleoli.
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http://dx.doi.org/10.1242/jcs.044461DOI Listing
March 2009