scholarly journals Cul4A and DDB1 Associate with Skp2 To Target p27Kip1 for Proteolysis Involving the COP9 Signalosome

2006 ◽  
Vol 26 (7) ◽  
pp. 2531-2539 ◽  
Author(s):  
Tanya Bondar ◽  
Anna Kalinina ◽  
Lyne Khair ◽  
Dragana Kopanja ◽  
Alo Nag ◽  
...  
Keyword(s):  
Decay Rate ◽  
Fission Yeast ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Cop9 Signalosome ◽  
A Subunit ◽  
Replication Inhibitor ◽  
F Box Protein ◽  
Interfering Rna

ABSTRACT DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.

scholarly journals Histone Deacetylase Inhibitors Reduce Steroidogenesis through SCF-Mediated Ubiquitination and Degradation of Steroidogenic Factor 1 (NR5A1)

2007 ◽  
Vol 27 (20) ◽  
pp. 7284-7290 ◽  
Author(s):  
Wei-Yi Chen ◽  
Jui-Hsia Weng ◽  
Chen-Che Huang ◽  
Bon-chu Chung
Keyword(s):  
Histone Deacetylase ◽  
Ubiquitin Ligase ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Hdac Inhibitors ◽  
E3 Ubiquitin Ligase ◽  
Steroidogenic Factor 1 ◽  
A Subunit ◽  
F Box Protein ◽  
Interfering Rna

ABSTRACT Histone deacetylase (HDAC) inhibitors such as trichostatin A and valproic acid modulate transcription of many genes by inhibiting the activities of HDACs, resulting in the remodeling of chromatin. Yet this effect is not universal for all genes. Here we show that HDAC inhibitors suppressed the expression of steroidogenic gene CYP11A1 and decreased steroid secretion by increasing the ubiquitination and degradation of SF-1, a factor important for the transcription of all steroidogenic genes. This was accompanied by increased expression of Ube2D1 and SKP1A, an E2 ubiquitin conjugase and a subunit of the E3 ubiquitin ligase in the Skp1/Cul1/F-box protein (SCF) family, respectively. Reducing SKP1A expression with small interfering RNA resulted in recovery of SF-1 levels, demonstrating that the activity of SCF E3 ubiquitin ligase is required for the SF-1 degradation induced by HDAC inhibitors. Overexpression of exogenous SF-1 restored steroidogenic activities even in the presence of HDAC inhibitors. Thus, increased SF-1 degradation is the cause of the reduction in steroidogenesis caused by HDAC inhibitors. The increased SKP1A expression and SCF-mediated protein degradation could be the mechanism underlying the mode of action of HDAC inhibitors.

scholarly journals A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers

2021 ◽  
Author(s):  
Floris Foijer ◽  
Lin Zhou ◽  
Fernando R Rosas Bringas ◽  
Bjorn Bakker ◽  
Judith E Simon ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Chemotherapy Resistance ◽  
Synthetic Lethal ◽  
Ubiquitin Ligase Complex ◽  
Synthetic Lethal Interactions ◽  
A Subunit ◽  
Scf Ubiquitin Ligase

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the SCF ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work might provide a new angle of how to exploit MELK overexpression in cancers and might thus lead to novel intervention strategies.

scholarly journals A Role for Fis1 in Both Mitochondrial and Peroxisomal Fission in Mammalian Cells

2005 ◽  
Vol 16 (11) ◽  
pp. 5077-5086 ◽  
Author(s):  
Annett Koch ◽  
Yisang Yoon ◽  
Nina A. Bonekamp ◽  
Mark A. McNiven ◽  
Michael Schrader
Keyword(s):  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Transmembrane Domain ◽  
Mitochondrial Fission ◽  
Ectopic Expression ◽  
Mammalian Homologue ◽  
Necessary And Sufficient ◽  
Interfering Rna ◽  
Peroxisome Division

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.

scholarly journals Overproduction of Polypeptides Corresponding to the Amino Terminus of the F-Box Proteins Cdc4p and Met30p Inhibits Ubiquitin Ligase Activities of Their SCF Complexes

2003 ◽  
Vol 2 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Cheryl Dixon ◽  
Lee Ellen Brunson ◽  
Mary Margaret Roy ◽  
Dechelle Smothers ◽  
Michael G. Sehorn ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Cellular Responses ◽  
Amino Terminus ◽  
Variable Component ◽  
Amino Terminal ◽  
F Box Protein ◽  
Substrate Proteins

ABSTRACT Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo.

scholarly journals A Subunit of the Anaphase-Promoting Complex Is a Centromere-Associated Protein in Mammalian Cells

1998 ◽  
Vol 18 (1) ◽  
pp. 468-476 ◽  
Author(s):  
Pia-Marie Jörgensen ◽  
Eva Brundell ◽  
Maria Starborg ◽  
Christer Höög
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Separation Process ◽  
Soluble Form ◽  
Anaphase Promoting Complex ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Ubiquitin Ligase Complex ◽  
A Subunit

ABSTRACT Sister chromatids in early mitotic cells are held together mainly by interactions between centromeres. The separation of sister chromatids at the transition between the metaphase and the anaphase stages of mitosis depends on the anaphase-promoting complex (APC), a 20S ubiquitin-ligase complex that targets proteins for destruction. A subunit of the APC, called APC-α in Xenopus (and whose homologs are APC-1, Cut4, BIME, and Tsg24), has recently been identified and shown to be required for entry into anaphase. We now show that the mammalian APC-α homolog, Tsg24, is a centromere-associated protein. While this protein is detected only during the prophase to the anaphase stages of mitosis in Chinese hamster cells, it is constitutively associated with the centromeres in murine cells. We show that there are two forms of this protein in mammalian cells, a soluble form associated with other components of the APC and a centromere-bound form. We also show that both the Tsg24 protein and the Cdc27 protein, another APC component, are bound to isolated mitotic chromosomes. These results therefore support a model in which the APC by ubiquitination of a centromere protein regulates the sister chromatid separation process.

scholarly journals Fission Yeast F-box Protein Pof3 Is Required for Genome Integrity and Telomere Function

2002 ◽  
Vol 13 (1) ◽  
pp. 211-224 ◽  
Author(s):  
Satoshi Katayama ◽  
Kenji Kitamura ◽  
Anna Lehmann ◽  
Osamu Nikaido ◽  
Takashi Toda
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Ubiquitin Ligase ◽  
Repeat Motif ◽  
High Rate ◽  
Genome Integrity ◽  
Gene Encoding ◽  
Cell Cycle Delay ◽  
C Terminus ◽  
F Box Protein

The Skp1-Cullin-1/Cdc53-F-box protein (SCF) ubiquitin ligase plays an important role in various biological processes. In this enzyme complex, a variety of F-box proteins act as receptors that recruit substrates. We have identified a fission yeast gene encoding a novel F-box protein Pof3, which contains, in addition to the F-box, a tetratricopeptide repeat motif in its N terminus and a leucine-rich-repeat motif in the C terminus, two ubiquitous protein–protein interaction domains. Pof3 forms a complex with Skp1 and Pcu1 (fission yeast cullin-1), suggesting that Pof3 functions as an adaptor for specific substrates. In the absence of Pof3, cells exhibit a number of phenotypes reminiscent of genome integrity defects. These include G2 cell cycle delay, hypersensitivity to UV, appearance of lagging chromosomes, and a high rate of chromosome loss.pof3 deletion strains are viable because the DNA damage checkpoint is continuously activated in the mutant, and this leads to G2 cell cycle delay, thereby preventing the mutant from committing lethal mitosis. Pof3 localizes to the nucleus during the cell cycle. Molecular analysis reveals that in this mutant the telomere is substantially shortened and furthermore transcriptional silencing at the telomere is alleviated. The results highlight a role of the SCFPof3 ubiquitin ligase in genome integrity via maintaining chromatin structures.

scholarly journals A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers

2021 ◽  
Author(s):  
Lin Zhou ◽  
Siqi Zheng ◽  
Fernando R Rosas Bringas ◽  
Bjorn Bakker ◽  
Judith E Simon ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Chemotherapy Resistance ◽  
Synthetic Lethal ◽  
Ubiquitin Ligase Complex ◽  
Synthetic Lethal Interactions ◽  
Histone Deacetylase 4 ◽  
A Subunit

Abstract Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the Skp, Cullin, F-box containing (SCF) ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (Histone Deacetylase 4, HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work identified a novel potential drug target for tumors that overexpress MELK.

scholarly journals Null Mutation of AtCUL1 Causes Arrest in Early Embryogenesis in Arabidopsis

2002 ◽  
Vol 13 (6) ◽  
pp. 1916-1928 ◽  
Author(s):  
Wen-Hui Shen ◽  
Yves Parmentier ◽  
Hanjo Hellmann ◽  
Esther Lechner ◽  
Aiwu Dong ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Critical Role ◽  
Functional Characterization ◽  
Early Embryogenesis ◽  
Cell Cycle Regulators ◽  
Genetic Studies ◽  
Scf Ubiquitin Ligase ◽  
F Box Protein

The SCF (for SKP1, Cullin/CDC53,F-box protein) ubiquitin ligase targets a number of cell cycle regulators, transcription factors, and other proteins for degradation in yeast and mammalian cells. Recent genetic studies demonstrate that plant F-box proteins are involved in auxin responses, jasmonate signaling, flower morphogenesis, photocontrol of circadian clocks, and leaf senescence, implying a large spectrum of functions for the SCF pathway in plant development. Here, we present a molecular and functional characterization of plant cullins. TheArabidopsis genome contains 11 cullin-related genes. Complementation assays revealed that AtCUL1 but not AtCUL4 can functionally complement the yeast cdc53 mutant.Arabidopsis mutants containing transfer DNA (T-DNA) insertions in the AtCUL1 gene were shown to display an arrest in early embryogenesis. Consistently, both the transcript and the protein of the AtCUL1 gene were found to accumulate in embryos. The AtCUL1 protein localized mainly in the nucleus but also weakly in the cytoplasm during interphase and colocalized with the mitotic spindle in metaphase. Our results demonstrate a critical role for the SCF ubiquitin ligase inArabidopsis embryogenesis.

scholarly journals Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination

2021 ◽  
Author(s):  
Jie Su ◽  
Naoko Toyofuku ◽  
Takuro Nakagawa
Keyword(s):  
Fission Yeast ◽  
Ubiquitin Ligase ◽  
Inverted Repeat ◽  
Chromosomal Rearrangements ◽  
Ring Finger ◽  
Sliding Clamp ◽  
Gross Chromosomal Rearrangements ◽  
Ubiquitin Ligase Activity

AbstractRad52 recombinase can cause gross chromosomal rearrangements (GCRs). However, the mechanism of Rad52-dependent GCRs remains unclear. Here, we show that fission yeast Rad8/HLTF facilitates Rad52-dependent GCRs through the ubiquitination of lysine 107 (K107) of PCNA, a DNA sliding clamp. Loss of Rad8 reduced isochromosomes resulting from centromere inverted repeat recombination. Rad8 HIRAN and RING finger mutations reduced GCRs, suggesting that Rad8 facilitates GCRs through 3’ DNA-end binding and ubiquitin ligase activity. Mms2 and Ubc4 but not Ubc13 ubiquitin-conjugating proteins were required for GCRs. Consistent with this, PCNA K107R but not K164R mutation greatly reduced GCRs. Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs, as PCNA K107R, rad8, and rad52 mutations epistatically reduced GCRs. Remarkably, K107 is located at the interface between PCNA subunits, and an interface mutation D150E bypassed the requirement of PCNA K107 ubiquitination for GCRs. This study uncovers the role of Rad8-dependent PCNA K107 ubiquitination in Rad52-dependent GCRs.

scholarly journals TAZ Promotes PC2 Degradation through a SCFβ-Trcp E3 Ligase Complex

2007 ◽  
Vol 27 (18) ◽  
pp. 6383-6395 ◽  
Author(s):  
Yu Tian ◽  
Robert Kolb ◽  
Jeong-Ho Hong ◽  
John Carroll ◽  
Dawei Li ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Transcriptional Regulator ◽  
Degradation Pathway ◽  
Cystic Kidney ◽  
Vertebrate Species ◽  
Polycystic Kidney ◽  
Binding Partner ◽  
F Box Protein

ABSTRACT Studies of a TAZ knockout mouse reveal a novel function of the transcriptional regulator TAZ, that is, as a binding partner of the F-box protein β-Trcp. TAZ−/− mice develop polycystic kidney disease (PKD) and emphysema. The calcium-permeable cation channel protein polycystin 2 (PC2) is overexpressed in kidneys of TAZ−/− mice as a result of decreased degradation via an SCFβ-Trcp E3 ubiquitin ligase pathway. Replacements of serines in a phosphodegron motif in TAZ prevent β-Trcp binding and PC2 degradation. Coexpression of a cytoplasmic fragment of polycystin 1 blocks the PC2-TAZ interaction and prevents TAZ-mediated degradation of PC2. Depletion of TAZ in zebrafish also results in a cystic kidney accompanied by overexpression of PC2. These results establish a common role of TAZ across vertebrate species in a protein degradation pathway regulated by phosphorylation and implicate deficiencies in this pathway in the development of PKD.

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