Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2

Kaiser, Peter; Mandl, Sonja; Schweiger, Manfred; Schneider, Rainer

doi:10.1016/0014-5793(95)01323-7

Cited by 19 publications

(21 citation statements)

References 32 publications

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“…In E2 enzymes, the core Ubc domain appears to be sufficient for their enzymatic activity, while the extensions at either end could serve as modules for protein-protein interactions that direct the enzyme to specific substrates or subcellular locations (2,20,27,38,48). The unique amino termini of the different isoforms of HsUEV-1 might be involved in specific protein-protein interactions which may affect their localization or activity.…”

Section: Discussionmentioning

confidence: 99%

Role of UEV-1, an Inactive Variant of the E2 UbiquitinConjugating Enzymes, in In Vitro Differentiation and Cell Cycle Behavior of HT-29-M6 Intestinal Mucosecretory Cells

Sancho¹,

Vilà²,

Sánchez‐Pulido

et al. 1998

Molecular and Cellular Biology

133

137

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By means of differential RNA display, we have isolated a cDNA corresponding to transcripts that are down-regulated upon differentiation of the goblet cell-like HT-29-M6 human colon carcinoma cell line. These transcripts encode proteins originally identified as CROC-1 on the basis of their capacity to activate transcription of c-fos. We show that these proteins are similar in sequence, and in predicted secondary and tertiary structure, to the ubiquitin-conjugating enzymes, also known as E2. Despite the similarities, these proteins lack a critical cysteine residue essential for the catalytic activity of E2 enzymes and, in vitro, they do not conjugate or transfer ubiquitin to protein substrates. These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeasts to mammals. Therefore, we have designated them UEV (ubiquitin-conjugating E2 enzyme variant) proteins, defined as proteins similar in sequence and structure to the E2 ubiquitin-conjugating enzymes but lacking their enzymatic activity (HW/GDB-approved gene symbol, UBE2V). At least two human genes code for UEV proteins, and one of them, located on chromosome 20q13.2, is expressed as at least four isoforms, generated by alternative splicing. All human cell types analyzed expressed at least one of these isoforms. Constitutive expression of exogenous human UEV in HT-29-M6 cells inhibited their capacity to differentiate upon confluence and caused both the entry of a larger proportion of cells in the division cycle and an accumulation in G 2 -M. This was accompanied with a profound inhibition of the mitotic kinase, cdk1. These results suggest that UEV proteins are involved in the control of differentiation and could exert their effects by altering cell cycle distribution.The intestinal epithelium is comprised of cells with different mature phenotypes that are believed to derive from common precursor cells resident in special anatomic compartments, called the crypts of Lieberkühn. Through asymmetric divisions and migration along the crypt, such precursor cells undergo phenotypic conversion into mucosecretory, absorptive, enteroendocrine or Paneth cells (19), with each expressing a distinct set of molecules characteristic of their specialized mature functions. The life cycle of mature cells terminates by apoptosis, followed by shedding from the tip of the villus to the intestinal lumen (19).A number of cellular models, such as the human colorectal cancer-derived cell lines HT-29-M6, HS174T, and Caco-2, have allowed the analysis of the molecular mechanisms that control the differentiated phenotypes of human intestinal epithelial cells (24,36,44,47,73). Several of these models appear to recapitulate some of the differentiation processes that accompany developmentally regulated events, such as the establishment of cephalocaudal and crypt-villus axes (59, 62). Using either in vitro or in vivo models, systematic approaches have led to the identification of differentially expressed genes and proteins involved in th...

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Section: Discussionmentioning

confidence: 99%

Role of UEV-1, an Inactive Variant of the E2 UbiquitinConjugating Enzymes, in In Vitro Differentiation and Cell Cycle Behavior of HT-29-M6 Intestinal Mucosecretory Cells

Sancho¹,

Vilà²,

Sánchez‐Pulido

et al. 1998

Molecular and Cellular Biology

133

137

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“…No GSTubiquitin was transferred to HERC5 under these experimental conditions, indicating that the E2 enzymes present in the RRL were not able to transfer ubiquitin to HERC5. Therefore, the E2 enzymes H3, H5a, H5b, H5c, H6, H7, H8 and H10 were added to the assay (Jensen et al, 1995;Kaiser et al, 1995;Kumar et al, 1997;Nuber et al, 1996;Plon et al, 1993;Scheffner et al, 1994;Townsley et al, 1997). E2 enzyme activity was controlled as described in Materials and Methods.…”

Section: E3 Ubiquitin Ligase Activity Of Herc5mentioning

confidence: 99%

HERC5, a HECT E3 ubiquitin ligase tightly regulated in LPS activated endothelial cells

Kroismayr

Baranyi

Stehlik

et al. 2004

Journal of Cell Science

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By differential screening we isolated genes upregulated in inflammatory cytokine-stimulated human skin microvascular endothelial cells. One of these cDNAs encoded RCC1 (regulator of chromosome condensation 1)-like repeats and a HECT (homologous to E6-AP C-terminus) domain, representing a member of the HERC (HECT and RCC1 domain protein) family of ubiquitin ligases. The mRNA level of this member, HERC5, is specifically upregulated in endothelial cells by the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β, and by lipopolysaccharide (LPS), but is hardly expressed in other cells of the vascular wall such as primary smooth muscle cells and fibroblasts. Regulation of HERC5 gene expression suggests a critical role for the transcription factor NF-κB. In contrast to mRNA expression HERC5 protein is subject of enhanced degradation upon LPS stimulation of endothelial cells. The time course of LPS-induced changes in HERC5 protein and mRNA levels suggests that the initial drop in HERC5 protein is balanced by increased protein synthesis due to upregulation of HERC5 mRNA. This leads to recovery of HERC5 protein levels within 12 hours of LPS stimulation and points at a tight control of HERC5 protein. To analyze functional activity of this putative member of the ubiquitin-conjugating pathway we performed in vitro assays with different ubiquitin-conjugating enzymes. We found that HERC5 possesses ubiquitin ligase activity and requires the presence of the ubiquitin-conjugating enzyme UbcH5a for its activity. These data show for the first time that a functionally active HECT ubiquitin ligase exhibits a tightly controlled cytosolic level under inflammatory conditions in endothelial cells.

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“…The enzymes that attach ubiquitin to protein substrates, known as ubiquitin-conjugating enzymes, share a common active site and have unique domains that determine substrate specificity and intracellular localization (11)(12)(13); it is likely that ubiquitin-processing enzymes have similar properties, but, so far, none have been defined.…”

Section: Introductionmentioning

confidence: 99%

A mutant deubiquitinating enzyme (Ubp-M) associates with mitotic chromosomes and blocks cell division

Cai

Babbitt

Marchesi

1999

Proc. Natl. Acad. Sci. U.S.A.

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A BSTR ACTA new ubiquitin-processing protease (Ubp-M) has been identified in mammalian cells that is phosphorylated at the onset of mitosis and dephosphorylated during the metaphase͞anaphase transition. The carboxylterminal domain of this 823-aa protein can be phosphorylated in vitro with either extracts of mitotic cells or purified cdc-2͞ cyclin B complexes. Recombinant Ubp-M is able to deubiquitinate histone H2A in vitro, and the phosphorylated form is also enzymatically active. Wild-type Ubp-M, transiently expressed as green f luorescent protein-fusion proteins, localizes in the cytoplasm of cultured cells, but mutant forms, lacking an active-site cysteine, associate closely with mitotic chromosomes during all stages of cell division and remain within the nucleus during the postmitotic period. Cells transfected with plasmids containing mutant Ubp-M genes stop dividing and eventually undergo apoptosis. Ubp-M may deubiquitinate one or more critical proteins that are involved in the condensation of mitotic chromosomes, possibly acting selectively on histones H2A and H2B, the major ubiquitinated proteins of chromatin.A large number of critical regulatory proteins are modified covalently by the enzymatic attachment of ubiquitin to side chains of lysines, a modification that often leads to their proteolytic degradation (1, 2). Proteins that regulate the cell cycle, such as cyclin B and p27, and either activate or inhibit specific protein kinases are ubiquitinated by multiprotein complexes, known as the anaphase-promoting complexes or cyclosomes (3, 4).It is generally accepted that the covalently bound polyubiquitin chains, linked together as polymers through internal isopeptide bonds, are recognized by one or more subunits of the 26S proteosome as the first step in the pathway that eventually leads to degradation of the ubiquitinated protein (5-7). Although the ubiquitins themselves are 76-aa polypeptides, they are not degraded by the proteosomes, but instead are recycled through the actions of enzymes known as ubiquitin-processing proteases (Ubps) or deubiquitinating enzymes (DUBs). The enzymes and protein cofactors that attach ubiquitin to polypeptides have been studied extensively (8, 9), but our understanding of how the ubiquitin-processing enzymes function lags far behind. A number of proteins are ubiquitinated and then degraded during mitosis, but it is not known how, when, or where deubiquitinating enzymes act during the mitotic cycle. Furthermore, many studies indicate that the recycling of ubiquitin from degraded proteins by deubiquitinating enzymes is not their sole function.Yeast are believed to have 20 or more proteins that may have ubiquitin-processing activity (10), but it remains unresolved how individual Ubps recognize specific substrates or how they themselves are regulated. The enzymes that attach ubiquitin to protein substrates, known as ubiquitin-conjugating enzymes, share a common active site and have unique domains that determine substrate specificity and intracellular localization (11-13); it...

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Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2

Cited by 19 publications

References 32 publications

Role of UEV-1, an Inactive Variant of the E2 UbiquitinConjugating Enzymes, in In Vitro Differentiation and Cell Cycle Behavior of HT-29-M6 Intestinal Mucosecretory Cells

Role of UEV-1, an Inactive Variant of the E2 UbiquitinConjugating Enzymes, in In Vitro Differentiation and Cell Cycle Behavior of HT-29-M6 Intestinal Mucosecretory Cells

HERC5, a HECT E3 ubiquitin ligase tightly regulated in LPS activated endothelial cells

A mutant deubiquitinating enzyme (Ubp-M) associates with mitotic chromosomes and blocks cell division

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