The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

ABSTRACT The yeast CTDK-I complex has been implicated in phosphorylation of the carboxy-terminal domain of the RNA polymerase II and in transcription control. It is composed of three polypeptides: Ctk1p and Ctk2p, a cyclin-dependent kinase and a C-type cyclin subunit, respectively; and Ctk3p, a third subunit of unknown function. Cyclins are regulatory proteins whose expression is tightly controlled at the protein level. In this study, we examined the regulation of Ctk2p expression in vivo. Surprisingly, unlike what has been described for cell cycle cyclins, steady-state levels of Ctk2p are composed of two relatively abundant forms, one of them phosphorylated. We show that this phosphorylated form is extremely unstable (half-life, 5 min) and that rapid proteolysis of Ctk2p exhibits growth-related regulation. Furthermore, our data establish that similar to the case for other naturally short-lived proteins, Ctk2p degradation is mediated by the ubiquitin-proteasome pathway. This is the first demonstration that a C-type cyclin is phosphorylated and targeted to the proteasome. Strikingly, neither phosphorylation nor destruction of Ctk2p requires its associated kinase Ctk1p, a feature fundamentally different from that which has been observed for cell cycle cyclins.

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The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner

Journal of Cell Science ◽

10.1242/jcs.113.23.4363 ◽

2000 ◽

Vol 113 (23) ◽

pp. 4363-4371 ◽

Cited By ~ 3

Author(s):

J. Zhao ◽

T. Tenev ◽

L.M. Martins ◽

J. Downward ◽

N.R. Lemoine

Keyword(s):

Cell Cycle ◽

Proteasome Inhibitors ◽

Dependent Manner ◽

Ubiquitin Proteasome Pathway ◽

Apoptosis Protein ◽

Ubiquitin Proteasome ◽

A Cell ◽

Proteasome Pathway ◽

Cycle Dependent

Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G(2)/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G(1) phase. It has been suggested that cell cycle-dependent expression of survivin is regulated at the transcriptional level. In this study we demonstrate involvement of the ubiquitin-proteasome pathway in post-translational regulation of survivin. Survivin is a short-lived protein with a half-life of about 30 minutes and proteasome inhibitors greatly stabilise survivin in vivo. Expression of the survivin gene under the control of the CMV promoter cannot block cell cycle-dependent degradation of the protein. Proteasome inhibitors can block survivin degradation during the G(1) phase and polyubiquitinated derivatives can be detected in vivo. Mutation of critical amino acid residues within the baculovirus IAP repeat (BIR) domain or truncation of the N terminus or the C terminus sensitises survivin to proteasome degradation. Together, these results indicate that the ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner and structural changes greatly destabilise the survivin protein.

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The RING Domain of cIAP1 Mediates the Degradation of RING-bearing Inhibitor of Apoptosis Proteins by Distinct Pathways

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0107 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2729-2740 ◽

Cited By ~ 42

Author(s):

Herman H. Cheung ◽

Stéphanie Plenchette ◽

Chris J. Kern ◽

Douglas J. Mahoney ◽

Robert G. Korneluk

Keyword(s):

Fas Ligand ◽

Ring Domain ◽

Inhibitor Of Apoptosis ◽

Inhibitor Of Apoptosis Proteins ◽

Down Regulation ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Apoptosis Proteins

The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR–mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.

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S-Nitrosylation of IRP2 Regulates Its Stability via the Ubiquitin-Proteasome Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.330-337.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 330-337 ◽

Cited By ~ 64

Author(s):

Sangwon Kim ◽

Simon S. Wing ◽

Prem Ponka

Keyword(s):

Regulatory Protein ◽

Untranslated Regions ◽

Raw 264.7 Cells ◽

Ubiquitin Proteasome Pathway ◽

Functional Implications ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Iron Responsive Elements

ABSTRACT Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.

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c-Myc Proteolysis by the Ubiquitin-Proteasome Pathway: Stabilization of c-Myc in Burkitt's Lymphoma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.20.7.2423-2435.2000 ◽

2000 ◽

Vol 20 (7) ◽

pp. 2423-2435 ◽

Cited By ~ 272

Author(s):

Mark A. Gregory ◽

Stephen R. Hann

Keyword(s):

Amino Acids ◽

Phosphorylation Site ◽

Burkitt’S Lymphoma ◽

Burkitt's Lymphoma ◽

Lymphoma Cells ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Burkitt’S Lymphoma Cells

ABSTRACT The c-Myc oncoprotein is a transcription factor which is a critical regulator of cellular proliferation. Deregulated expression of c-Myc is associated with many human cancers, including Burkitt's lymphoma. The c-Myc protein is normally degraded very rapidly with a half-life of 20 to 30 min. Here we demonstrate that proteolysis of c-Myc in vivo is mediated by the ubiquitin-proteasome pathway. Inhibition of proteasome activity blocks c-Myc degradation, and c-Myc is a substrate for ubiquitination in vivo. Furthermore, an increase in c-Myc stability occurs in mitotic cells and is associated with inhibited c-Myc ubiquitination. Deletion analysis was used to identify regions of the c-Myc protein which are required for rapid proteolysis. We found that a centrally located PEST sequence, amino acids 226 to 270, is necessary for rapid c-Myc degradation, but not for ubiquitination. Also, N-terminal sequences, located within the first 158 amino acids of c-Myc, are necessary for both efficient c-Myc ubiquitination and subsequent degradation. We found that c-Myc is significantly stabilized (two- to sixfold) in many Burkitt's lymphoma-derived cell lines, suggesting that aberrant c-Myc proteolysis may play a role in the pathogenesis of Burkitt's lymphoma. Finally, mutation of Thr-58, a major phosphorylation site in c-Myc and a mutational hot spot in Burkitt's lymphoma, increases c-Myc stability; however, mutation of c-Myc is not essential for stabilization in Burkitt's lymphoma cells.

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An Ultra-High Throughput Cell-Based Screen for Wee1 Degradation Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110375848 ◽

2010 ◽

Vol 15 (8) ◽

pp. 907-917 ◽

Cited By ~ 11

Author(s):

Franck Madoux ◽

Scott Simanski ◽

Peter Chase ◽

Jitendra K. Mishra ◽

William R. Roush ◽

...

Keyword(s):

Cell Cycle ◽

Fusion Protein ◽

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Cell Cycle Progression ◽

Ubiquitin Proteasome Pathway ◽

Subsequent Degradation ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-luciferase fusion protein. To ensure ultra-high-throughput screening (uHTS) compatibility, the assay was scaled to a 1536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogeneous assay demonstrated robust performance, with a calculated Z′ factor of 0.65 ± 0.05. The assay was screened against a publicly available library of ~218,000 compounds to identify Wee1 stabilizers. Nonselective, cytotoxic, and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of an N-cyclin B-luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of 4 unrelated cell-permeable small molecules that showed selective Wee1-luciferase stabilization with micromolar potency. One of these compounds, SID4243143 (ML 118), was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. Results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.

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The importance of ubiquitin E3 ligases, SCF and APC/C, in human cancers

Medicine and Pharmacy Reports ◽

10.15386/cjmed-377 ◽

2015 ◽

Vol 88 (1) ◽

pp. 9-14 ◽

Cited By ~ 4

Author(s):

Ovidiu Vasile Bochis ◽

Bogdan Fetica ◽

Catalin Vlad ◽

Patriciu Achimas-Cadariu ◽

Alexandru Irimie

Keyword(s):

Cell Cycle ◽

Anaphase Promoting Complex ◽

E3 Ligases ◽

Cyclin Dependent Kinases ◽

Ubiquitin Proteasome Pathway ◽

Complex Process ◽

Target Therapies ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

F Box Protein

A normal evolution of the cell-cycle phases consists of multiple consecutive events, which makes it a highly complex process. Its preservation is regulated by Cyclin-Cdks (cyclin-dependent kinases) interactions and protein degradation, which is often controlled by the ubiquitin-mediated proteolysis.The goal of this review is to emphasize the most important features of the regulation of the cell-cycle involved in cancerogenesis, by presenting the involvement of E3 ubiquitin ligases SCF (Skp1-Cul1-F-box protein) and APC/C (Anaphase-promoting complex/cyclosome) in human malignancies. Also, we discuss the importance of the ubiquitin proteasome pathway blockade in cancer treatment. We know that a better understanding of the regulatory biology of the cell cycle can lead to the development of new target therapies for cancer.

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Reproductive Cytotoxicity Is Predicted by Magnetic Resonance Microscopy and Confirmed by Ubiquitin–Proteasome Immunohistochemistry in a Theophylline-Induced Model of Rat Testicular and Epididymal Toxicity

Microscopy and Microanalysis ◽

10.1017/s143192760505021x ◽

2005 ◽

Vol 11 (4) ◽

pp. 300-312 ◽

Cited By ~ 7

Author(s):

M.W. Tengowski ◽

P. Sutovsky ◽

L.W. Hedlund ◽

D.J. Guyot ◽

J.E. Burkhardt ◽

...

Keyword(s):

Magnetic Resonance ◽

Phosphodiesterase Inhibitor ◽

Magnetic Resonance Microscopy ◽

Vascular Perfusion ◽

Ubiquitin Proteasome Pathway ◽

Organ Systems ◽

Water Signal ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

This study investigated the testicular changes in the rat induced by the nonspecific phosphodiesterase inhibitor, theophylline using magnetic resonance microscopy (MRM) and ubiquitin immunostaining techniques.In vivoT1- and T2-weighted images were acquired at 2 T under anesthesia. Increased signal observed in the theophylline-treated rats suggests that leakage of MRM contrast was occurring.In vivoMRM results indicate that day 16 testis displayed an increased T1-weighted water signal in the area of the seminiferous tubule that decreased by day 32. These findings were validated by histopathology, suggesting thatin vivoMRM has the sensitivity to predict changes in testis and epididymal tissues. The participation of the ubiquitin system was investigated, using probes for various markers of the ubiquitin-proteasome pathway. MRM can be used to detect subtle changes in the vascular perfusion of organ systems, and the up-regulation/mobilization of ubiquitin-proteasome pathway may be one of the mechanisms used in theophylline-treated epididymis to remove damaged cells before storage in the cauda epididymis. The combined use ofin vivoMRM and subsequent tissue or seminal analysis for the presence of ubiquitin in longitudinal studies may become an important biomarker for assessing testis toxicities drug studies.

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