Cell-cycle-dependent PC-PLC regulation by APC/CCdc20-mediated ubiquitin-proteasome pathway

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2527 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2527-2534 ◽

Cited By ~ 9

Author(s):

Guillaume Hautbergue ◽

Valérie Goguel

Keyword(s):

Cell Cycle ◽

Rna Polymerase Ii ◽

Cyclin Dependent Kinase ◽

Transcription Control ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Steady State Levels ◽

Proteasome Pathway ◽

Carboxy Terminal

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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Regulation of the ubiquitin proteasome pathway in human lens epithelial cells during the cell cycle

Experimental Eye Research ◽

10.1016/j.exer.2003.11.009 ◽

2004 ◽

Vol 78 (2) ◽

pp. 197-205 ◽

Cited By ~ 14

Author(s):

Qing Liu ◽

Fu Shang ◽

Weimin Guo ◽

Marisa Hobbs ◽

Paloma Valverde ◽

...

Keyword(s):

Cell Cycle ◽

Epithelial Cells ◽

Human Lens ◽

Lens Epithelial Cells ◽

Ubiquitin Proteasome Pathway ◽

Human Lens Epithelial Cells ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

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The Xeroderma Pigmentosum Group E Gene Product DDB2 Is a Specific Target of Cullin 4A in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.20.6738-6747.2001 ◽

2001 ◽

Vol 21 (20) ◽

pp. 6738-6747 ◽

Cited By ~ 106

Author(s):

Alo Nag ◽

Tanya Bondar ◽

Shalu Shiv ◽

Pradip Raychaudhuri

Keyword(s):

Cell Cycle ◽

Xeroderma Pigmentosum ◽

Mammalian Cells ◽

26S Proteasome ◽

Breast Cancers ◽

Ubiquitin Proteasome Pathway ◽

Specific Target ◽

Cullin 4A ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

ABSTRACT The damaged-DNA binding protein DDB consists of two subunits, DDB1 (127 kDa) and DDB2 (48 kDa). Mutations in the DDB2 subunit have been detected in patients suffering from the repair deficiency disease xeroderma pigmentosum (group E). In addition, recent studies suggested a role for DDB2 in global genomic repair. DDB2 also exhibits transcriptional activity. We showed that expression of DDB1 and DDB2 stimulated the activity of the cell cycle regulatory transcription factor E2F1. Here we show that DDB2 is a cell cycle-regulated protein. It is present at a low level in growth-arrested primary fibroblasts, and after release the level peaks at the G1/S boundary. The cell cycle regulation of DDB2 involves posttranscriptional mechanisms. Moreover, we find that an inhibitor of 26S proteasome increases the level of DDB2, suggesting that it is regulated by the ubiquitin-proteasome pathway. Our previous study indicated that the cullin family protein Cul-4A associates with the DDB2 subunit. Because cullins are involved in the ubiquitin-proteasome pathway, we investigated the role of Cul-4A in regulating DDB2. Here we show that DDB2 is a specific target of Cul-4A. Coexpression of Cul-4A, but not Cul-1 or other highly related cullins, increases the ubiquitination and the decay rate of DDB2. A naturally occurring mutant of DDB2 (2RO), which does not bind Cul-4A, is not affected by coexpression of Cul-4A. Studies presented here identify a specific function of the Cul-4A gene, which is amplified and overexpressed in breast cancers.

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