Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.2.19 (ubiquitin-protein ligase)
799 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. Ubiquitin-conjugating enzyme E2(25K) utilizes isolated ubiquitin as the substrate for synthesis of such chains, in which successive ubiquitin units are linked by isopeptide bonds involving the side chain of Lys-48 of one ubiquitin and the COOH group of Gly-76 of the next. During continuous synthesis of multiubiquitin chains in the presence of purified ubiquitin-activating enzyme and E2(25K), there was a slight discrimination against radioiodinated ubiquitin (2.3-fold reduction in specific radioactivity of diubiquitin relative to value expected for no discrimination). Single-turnover experiments employing stoichiometrically iodinated ubiquitin derivatives indicated that E2(25K) discriminates extremely strongly (greater than 20-fold reduction in kcat/Km for diubiquitin synthesis) against ubiquitin that is monoiodinated at Tyr-59. The modest overall selection effect observed in continuous reactions is in part due to the occurrence of discrimination only when iodotyrosylubiquitin is the acceptor (Lys-48 donor) in diubiquitin synthesis; iodotyrosylubiquitin is kinetically competent when it is the species being transferred to native ubiquitin. The competence as acceptor of a site-directed mutant form of ubiquitin bearing a Tyr to Phe substitution at position 59 indicated that discrimination against iodotyrosylubiquitin by E2(25K) is not due to loss of the hydrogen-bonding interactions of Tyr-59. Rather, iodotyrosylubiquitin may be unable to react with the ubiquitin adduct of E2(25K) for steric reasons. Discrimination against iodotyrosylubiquitin as acceptor is unique to E2(25K) among three enzymes surveyed: iodotyrosylubiquitin is a fully competent acceptor in diubiquitin synthesis catalyzed by E2(25K) and is also utilized for multiubiquitin chain synthesis by E2(14K) and ubiquitin-protein ligase. These findings should assist in the design of future studies concerning E2(25K) structure and function.
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PMID:Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K). 132 Nov 47

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.
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PMID:Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites. 334 27

Some receptor tyrosine kinases such as the receptors for epidermal-growth factor (EGF) and platelet-derived growth factor undergo polyubiquitination as a consequence of ligand binding. The EGF receptor is also ubiquitinated by treatment with herbimycin A, an ansamycin antibiotic widely used as a tyrosine kinase inhibitor. To investigate the mechanism of the receptor ubiquitination, we have established an assay system in which herbimycin-A-induced ubiquitination processes can be analyzed in vitro. We now show that herbimycin A treatment of the purified EGF receptor induces polyubiquitination of the receptor in rabbit-reticulocyte lysate. Both DEAE unadsorbed material (fraction I) and high salt eluate (fraction II) of the reticulocyte lysate are involved cooperatively in the ubiquitination process, where the ubiquitin-conjugating enzyme UBC4 can functionally substitute for fraction I. A ubiquitin-protein ligase-like activity, partially purified from fraction II by DEAE anion-exchange chromatography, also functions in concert with UBC4. The precise mechanism of herbimycin A-induced ubiquitination of the EGF receptor is not fully understood, however, our present findings suggest that direct interaction with herbimycin A results in some modification of the receptor which is recognized by the ubiquitin-conjugating system in rabbit-reticulocyte lysate.
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PMID:Identification of an ubiquitin-ligation system for the epidermal-growth-factor receptor--herbimycin A induces in vitro ubiquitination in rabbit-reticulocyte lysate. 928 47

Topoisomerase I (TOP1) relaxes superhelical DNA through a breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site as a transient intermediate. The antitumor drug camptothecin (CPT) and its analogs inhibit the rejoining step of the breakage/rejoining reaction, which traps the enzyme in covalent linkage with DNA (the cleavable complex). Little is known about the fate of cellular TOP1 trapped in the cleavable complex. We have analyzed TOP1 in mammalian cell lines treated with CPT. When CPT-treated cells were lysed with either SDS or alkali and analyzed by Western blotting, greater than 90% of the TOP1 was linked to DNA. Nuclease treatment of the cell lysate to remove the covalently linked DNA from TOP1 revealed a distinct ladder of higher molecular weight bands having properties indicative of multi-ubiquitin (Ub) conjugates of TOP1. Approximately 5-10% of TOP1 was present as these conjugates within minutes of CPT treatment. Consistent with ubiquitination, TOP1 was not modified in ts85 cells at the restrictive temperature for its thermolabile ubiquitin-activating enzyme (E1). Because conjugation with ubiquitin can mark proteins for destruction by the 26S proteasome, we analyzed TOP1 protein levels during prolonged CPT treatment. TOP1 protein levels were reduced to about 25% during CPT treatments of 2-4 h resulting from increased destruction, with the half-life dropping from 10-16 h down to 1-2 h. The destruction of TOP1, like the formation of Ub-TOP1 conjugates, was not observed in ts85 cells at the restrictive temperature. The destruction of TOP1 was also prevented in cells treated with MG-132 and lactacystin, specific inhibitors of the 26S proteasome. Finally, the multi-Ub conjugates of TOP1 were observed whether or not aphidicolin was included in cotreatment with CPT, indicating that replication fork activity was not involved in making TOP1 a substrate for ubiquitination. These results demonstrate that independent of DNA replication, the TOP1 cleavable complex is ubiquitinated and destroyed in cells treated with antitumor drugs that block the religation step of the TOP1 reaction.
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PMID:Ubiquitin-dependent destruction of topoisomerase I is stimulated by the antitumor drug camptothecin. 930 65

Ubiquitination of receptor protein-tyrosine kinases (RPTKs) terminates signaling by marking active receptors for degradation. c-Cbl, an adapter protein for RPTKs, positively regulates RPTK ubiquitination in a manner dependent on its variant SRC homology 2 (SH2) and RING finger domains. Ubiquitin-protein ligases (or E3s) are the components of ubiquitination pathways that recognize target substrates and promote their ligation to ubiquitin. The c-Cbl protein acted as an E3 that can recognize tyrosine-phosphorylated substrates, such as the activated platelet-derived growth factor receptor, through its SH2 domain and that recruits and allosterically activates an E2 ubiquitin-conjugating enzyme through its RING domain. These results reveal an SH2-containing protein that functions as a ubiquitin-protein ligase and thus provide a distinct mechanism for substrate targeting in the ubiquitin system.
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PMID:The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. 1057 87

This is my reminiscent essay of my research life, but not a review article of specific subject. We found in the 1960s that BCAs (the branched chain amino acids, valine, leucine, and isoleucine) are unique in being the least metabolized amino acids in liver due to low activity of their transaminase. Later it was found clinically that BCAs are quite effective for recovery from hepatic encephalopathy. Furthermore, they could restore protein metabolism by stimulating synthesis and inhibiting degradation of body proteins under stress conditions. The signal of BCAs seems to be mediated by the amino acid sensor, Ssyl, which induces the amino acid permease AGP1. After liver injury, hepatocytes regenerate actively. In the 1980s, to study the molecular mechanism involved, we used primary cultured rat hepatocytes, the gene expressions of which respond very well to nutrients and hormones in the medium and to cell density. We identified HGF (hepatocyte growth factor) as a potent mitogen. The HGF receptor is cMet, an oncogene, and it initiates tyrosine phosphorylation in cellular signal transduction. The proteasome is a unique protease consisting of a very large multisubunit complex, which shows energy- and ubiquitin-dependent activity. In the 1990s we characterized the molecular structures of its subunits. Recently, proteasomes were found to degrade the HGF receptor, cMet. Furthermore, the Grrlp transcription factor, which is stimulated by Ssyl described above, has been identified as a ubiquitin-protein ligase. These studies on BCA, HGF, and proteasomes seemed to be unrelated to each other when I was working, but recent studies have shown that they are very closely related. So I would like to discuss the relations of my old work to recent findings.
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PMID:BCA, HGF, and proteasomes. 1060 2

Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. LMP2A functions to downregulate B-cell signal transduction and viral reactivation from latency in EBV-immortalized B cells in vitro, and acts to provide B cells with both a survival and developmental signal in vivo. Identification of proteins associated with LMP2A is important for elucidation of the mechanism that LMP2A employs to regulate B-cell signal transduction and EBV latency. LMP2A is constitutively tyrosine phosphorylated and is associated with protein tyrosine kinases such as Lyn and Syk when specific LMP2A tyrosines are phosphorylated. The amino-terminal domain of LMP2A includes multiple proline-rich regions, which may provide binding sites for proteins containing SH3 or WW domains. In this study, we demonstrate that four cellular proteins bind specifically to two PPPPY (PY) motifs present within the LMP2A amino-terminal domain. Protein microsequence analysis determined that three of these proteins were AIP4, WWP2/AIP2, and Nedd4. All of these proteins are members of the Nedd4-like ubiquitin-protein ligases family and have conserved domains including the C2, WW, and ubiquitin-protein ligase domain. The mutation of both PY motifs completely abolished binding activity of these proteins to LMP2A and the interaction of AIP4 and WWP2 with LMP2A was confirmed in cell lines expressing LMP2A, WWP2, and AIP4. Furthermore, a reduction in the level of Lyn and the rapid turnover of LMP2A and Lyn were observed in LMP2A-expressing cells. These findings suggest that LMP2A recruits Nedd4-like ubiquitin-protein ligases and B-cell signal transduction molecules, resulting in the degradation of LMP2A and Lyn by a ubiquitin-dependent mechanism. This provides a new means by which LMP2A may modulate B-cell signal transduction.
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PMID:The Epstein-Barr virus latent membrane protein 2A PY motif recruits WW domain-containing ubiquitin-protein ligases. 1068 40

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.
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PMID:Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II. 1078 4

SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin-protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss of sli-1 but not of another negative regulator, ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin-ligase domain.
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PMID:Requirements of multiple domains of SLI-1, a Caenorhabditis elegans homologue of c-Cbl, and an inhibitory tyrosine in LET-23 in regulating vulval differentiation. 1107 24

The colony-stimulating factor-1 (CSF-1) receptor is a protein-tyrosine kinase that regulates the proliferation and differentiation of monocyte and macrophage precursors. Binding of CSF-1 to its receptor results in activation of the kinase domain and autophosphorylation on a number of tyrosine residues. Phosphorylated tyrosine residues function as binding sites for SH2 domain-containing signaling proteins. It is known that activated receptors are internalized and degraded, but the mechanics of this process remain largely unknown. Recently, evidence has started to emerge that the ubiquitin-protein ligase c-Cbl is involved in CSF-1 receptor degradation. In addition, there is evidence that the CSF-1 receptor carboxy-terminus is involved in down regulation of the receptor. Here we show that the c-Cbl tyrosine kinase-binding (TKB) domain binds in vitro and in vivo to the CSF-1 receptor. Binding is dependent on the receptor's protein-kinase activity. Deletion of the carboxy-terminus or mutation of Tyr 973 blocks binding. We further provide evidence that the CSF-1 receptor's carboxy-terminus is a substrate for autophosphorylation. Our observations are consistent with a model in which receptor autophosphorylation at Tyr 973 creates a binding site for c-Cbl. Association of c-Cbl with the receptor leads to ubiquitination, followed by receptor degradation.
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PMID:C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor's carboxy-terminus. 1185 Aug 25


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