scholarly journals Human Cdc34 and Rad6B Ubiquitin-Conjugating Enzymes Target Repressors of Cyclic AMP-Induced Transcription for Proteolysis

1999 ◽  
Vol 19 (7) ◽  
pp. 5001-5013 ◽  
Author(s):  
Debananda Pati ◽  
Marvin L. Meistrich ◽  
Sharon E. Plon
Keyword(s):  
Cyclic Amp ◽  
Germ Cells ◽  
Mammalian Cells ◽  
Mitotic Cell ◽  
Dominant Negative ◽  
Testicular Development ◽  
Developmentally Regulated ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

ABSTRACT Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIγ, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIγ and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIγ- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B −/−) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1–Cullin–F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.

UbcH5A, a member of human E2 ubiquitin-conjugating enzymes, is closely related to SFT, a stimulator of iron transport, and is up-regulated in hereditary hemochromatosis

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3288-3293 ◽  
Author(s):  
Sven G. Gehrke ◽  
Hans-Dieter Riedel ◽  
Thomas Herrmann ◽  
Boris Hadaschik ◽  
Karin Bents ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Hereditary Hemochromatosis ◽  
Regulatory Region ◽  
Iron Chelation ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

Abstract SFT, a stimulator of iron (Fe) transport, has been described as a transmembrane protein that facilitates the uptake of ferrous and ferric iron in mammalian cells. This study was initiated to investigate the 5′ regulatory region of SFT and its role in the etiology of hereditary hemochromatosis. Sequence analyses of the putative 5′ regulatory region revealed that the SFT cDNA sequence corresponds to intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Further mRNA expression studies using a sequence-specific reverse transcriptase–polymerase chain reaction (RT-PCR) assay showed that UbcH5A is significantly up-regulated in the liver of iron-overloaded patients with hereditary hemochromatosis, as previously published for SFT. However, in vitro studies on HepG2 cells failed to demonstrate any significant UbcH5A regulation in response to iron loading or iron chelation. In conclusion, in vivo mRNA expression data previously obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary hemochromatosis remains to be elucidated further.

Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MATα2 repressor

Cell ◽  
1993 ◽  
Vol 74 (2) ◽  
pp. 357-369 ◽  
Author(s):  
Ping Chen ◽  
Phoebe Johnson ◽  
Thomas Sommer ◽  
Stefan Jentsch ◽  
Mark Hochstrasser
Keyword(s):  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Vivo Degradation

scholarly journals OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Cells ◽  
Conjugating Enzyme

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

scholarly journals Two Ubiquitin-Conjugating Enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, Have Distinct Functions for Ubiquitination of Mitotic Cyclin

2003 ◽  
Vol 23 (10) ◽  
pp. 3497-3505 ◽  
Author(s):  
Hiroaki Seino ◽  
Tsutomu Kishi ◽  
Hideo Nishitani ◽  
Fumiaki Yamao
Keyword(s):  
Fission Yeast ◽  
Ubiquitin Proteasome System ◽  
High Expression ◽  
Mitotic Cyclin ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
Mutant Cells ◽  
Conjugating Enzyme

ABSTRACT Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4 + cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11 + cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.

scholarly journals Overexpression of Bcl-w in the Testis Disrupts Spermatogenesis: Revelation of a Role of BCL-W in Male Germ Cell Cycle Control

2003 ◽  
Vol 17 (9) ◽  
pp. 1868-1879 ◽  
Author(s):  
Wei Yan ◽  
Jun-Xing Huang ◽  
Anna-Stina Lax ◽  
Lauri Pelliniemi ◽  
Eeva Salminen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Testicular Development ◽  
Dna Ladder

Abstract To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken β-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5′-bromo-2′deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.

scholarly journals A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

1997 ◽  
Vol 17 (12) ◽  
pp. 7362-7374 ◽  
Author(s):  
J A Diehl ◽  
C J Sherr
Keyword(s):  
Cyclin D1 ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Linker Peptide ◽  
Cdk Activating Kinase

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.

scholarly journals T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer

2005 ◽  
Vol 201 (9) ◽  
pp. 1367-1373 ◽  
Author(s):  
Bradley S. Cobb ◽  
Tatyana B. Nesterova ◽  
Elizabeth Thompson ◽  
Arnulf Hertweck ◽  
Eric O'Connor ◽  
...  
Keyword(s):  
T Cell ◽  
Mammalian Cells ◽  
Early Stage ◽  
Early Embryo ◽  
Specific Gene ◽  
Small Interfering Rnas ◽  
Developmentally Regulated ◽  
Micro Rnas ◽  
Wide Range

The ribonuclease III enzyme Dicer is essential for the processing of micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability, and translation in a wide range of organisms. To provide a model system to explore the role of Dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional Dicer allele. Deletion of Dicer at an early stage of T cell development compromised the survival of αβ lineage cells, whereas the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, Dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin), and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one third of mammalian mRNAs are putative miRNA targets, Dicer seems to be dispensable for CD4/8 lineage commitment, a process in which epigenetic regulation of lineage choice has been well documented. Thus, although Dicer seems to be critical for the development of the early embryo, it may have limited impact on the implementation of some lineage-specific gene expression programs.

scholarly journals Expression of Atp8b3 in murine testis and its characterization as a testis specific P-type ATPase

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 345-351 ◽  
Author(s):  
Eun-Yeung Gong ◽  
Eunsook Park ◽  
Hyun Joo Lee ◽  
Keesook Lee
Keyword(s):  
Germ Cells ◽  
Testicular Development ◽  
Sperm Function ◽  
Developmentally Regulated ◽  
Primary Amino Acid Sequence ◽  
Biochemical Characteristics ◽  
Cellular Processes ◽  
Complex Process ◽  
Primary Amino ◽  
P Type

Spermatogenesis is a complex process that produces haploid motile sperms from diploid spermatogonia through dramatic morphological and biochemical changes. P-type ATPases, which support a variety of cellular processes, have been shown to play a role in the functioning of sperm. In this study, we isolated one putative androgen-regulated gene, which is the previously reported sperm-specific aminophospholipid transporter (Atp8b3, previously known asSaplt), and explored its expression pattern in murine testis and its biochemical characteristics as a P-type ATPase.Atp8b3is exclusively expressed in the testis and its expression is developmentally regulated during testicular development. Immunohistochemistry of the testis reveals thatAtp8b3is expressed only in germ cells, especially haploid spermatids, and the protein is localized in developing acrosomes. As expected, from its primary amino acid sequence, ATP8B3 has an ATPase activity and is phosphorylated by an ATP-producing acylphosphate intermediate, which is a signature property of the P-Type ATPases. Together, ATP8B3 may play a role in acrosome development and/or in sperm function during fertilization.

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