Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of .beta.,.gamma.-nonhydrolyzable ATP analogs

Johnston, Nancy L.; Cohen, Robert E.

doi:10.1021/bi00244a021

Cited by 42 publications

(30 citation statements)

References 49 publications

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“…6A). Because ATP␥S can be used as an energy source by E1, substrates that are not signaled for conjugation by phosphorylation can be conjugated in the presence of ATP␥S as an energy source (41). In contrast, substrates that are targeted following phosphorylation, such as IB␣, are not conjugated in the presence of ATP␥S that cannot serve as a donor of a phosphate group.…”

Section: Discussionmentioning

confidence: 99%

“…5B). In conjugation assays, ATP␥S is commonly used as an energy source for the conjugation process because it can support E1-mediated ubiquitin activation but not proteasomal degradation (41). However, the nonhydrolyzable analog of ATP cannot support conjugation of substrates that must undergo phosphorylation to be recognized by the conjugation machinery.…”

Section: Fig 3 Lmp1 Is Ubiquitinated and Its Degradation Requires Amentioning

confidence: 99%

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Degradation of the Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) by the Ubiquitin-Proteasome Pathway

Aviel¹,

Winberg²,

Massucci³

et al. 2000

Journal of Biological Chemistry

160

115

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The latent membrane protein 1 (LMP1) of the EpsteinBarr virus is a constitutively active receptor essential for B lymphocyte transformation by the Epstein-Barr virus. It is a short-lived protein, but the proteolytic pathway involved in its degradation is not known. The ubiquitin pathway is a major system for specific protein degradation in eukaryotes. Most plasma membrane substrates of the pathway are internalized upon ubiquitination and delivered for degradation in the lysosome/ vacuole. Here we show that LMP1 is a substrate of the ubiquitin pathway and is ubiquitinated both in vitro and in vivo. However, in contrast to other plasma membrane substrates of the ubiquitin system, it is degraded mostly by the proteasome and not by lysosomes. Degradation is independent of the single Lys residue of the protein; a lysine-less mutant LMP1 is degraded in a ubiquitin-and proteasome-dependent manner similar to the wild type protein. Degradation of both wild type and lysine-less protein is sensitive to fusion of a Myc tag to the N terminus of LMP1. In addition, deletion of as few as 12 N-terminal amino acid residues stabilizes the protein. These findings suggest that the first event in LMP1 degradation is attachment of ubiquitin to the N-terminal residue of the protein. We present evidence suggesting that phosphorylation is also required for degradation of LMP1.

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Section: Discussionmentioning

confidence: 99%

Section: Fig 3 Lmp1 Is Ubiquitinated and Its Degradation Requires Amentioning

confidence: 99%

Degradation of the Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) by the Ubiquitin-Proteasome Pathway

Aviel¹,

Winberg²,

Massucci³

et al. 2000

Journal of Biological Chemistry

160

115

View full text Add to dashboard Cite

show abstract

“…To examine whether CD28-mediated down-regulation of p27 kip1 involves ubiquitin-targeted proteolysis, cultures of T cells with anti-CD3 and anti-CD28 were established in either media alone or in the presence of Ubal. Ubal is a potent and specific inhibitor of multiple ubiquitin hydrolases involved in pathways of intracellular protein ubiquitin-dependent modification and turnover and decreases the rate of ubiquitin-dependent degradation (57). The presence of Ubal prevented the down-regulation of p27 kip1 (Fig.…”

Section: Down-regulation Of P27 By Cd28 Costimulation Is Controlled Amentioning

confidence: 95%

CD28 Costimulation Mediates T Cell Expansion Via IL-2-Independent and IL-2-Dependent Regulation of Cell Cycle Progression

Appleman

Berezovskaya

Grass

et al. 2000

The Journal of Immunology

182

152

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In the presence of TCR ligation by Ag, CD28 pathway mediates the most potent costimulatory signal for T cell activation, cytokine secretion, and T cell expansion. Although CD28 costimulation promotes T cell expansion due to IL-2 secretion and subsequent signaling via the IL-2 receptor, recent studies indicate that the dramatic T cell expansion mediated through the unopposed CD28 stimulation in CTLA4-deficient mice is IL-2 independent. Therefore, we sought to dissect the effects of CD28 and IL-2 receptor pathways on cell cycle progression and determine the molecular mechanisms by which the CD28 pathway regulates T cell expansion. Here we show that CD28 costimulation directly regulates T cell cycle entry and progression through the G1 phase in an IL-2-independent manner resulting in activation of cyclin D2-associated cdk4/cdk6 and cyclin E-associated cdk2. Subsequent progression into the S phase is mediated via both IL-2-dependent and IL-2-independent mechanisms and, although in the absence of IL-2 the majority of T cells are arrested at the G1/S transition, a significant fraction of them progresses into the S phase. The key regulatory mechanism for the activation of cyclin-cdk complexes and cell cycle progression is the down-regulation of p27kip1 cdk inhibitor, which is mediated at the posttranscriptional level by its ubiquitin-dependent degradation in the proteasome pathway. Therefore, CD28 costimulation mediates T cell expansion in an IL-2-independent and IL-2 dependent manner and regulates cell cycle progression at two distinct points: at the early G1 phase and at the G1/S transition.

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“…The conjugating system contained either ATP␥S or ATP (see "Experimental Procedures"). The ATP analog is utilized in conjugation assays since it promotes conjugation (via activation of E1), but inhibits degradation (since the 26 S proteasome can utilize only ATP), and therefore leads to accumulation of ubiquitin adducts (42). We were surprised to find that the nonhydrolyzable nucleotide analog is active, although not more than ATP, in promoting Hsc70-and cation-dependent conjugation that is apparently dependent on ATP hydrolysis.…”

Section: Gapdh and Rcm-␣-lamentioning

confidence: 99%

Ubiquitin-dependent Degradation of Certain Protein Substrates in Vitro Requires the Molecular Chaperone Hsc70

Bercovich

Stancovski

Mayer

et al. 1997

Journal of Biological Chemistry

275

177

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Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin and degradation of the tagged substrate. Conjugation is catalyzed via a three-step mechanism that involves three distinct enzymes that act successively: E1, E2, and E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate is specifically bound, catalyzes formation of a polyubiquitin chain that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we demonstrate that this protein is required for the degradation of actin, ␣-crystallin, glyceraldehyde-3-phosphate dehydrogenase, ␣-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is Hsc70-independent. Mechanistic analysis revealed that the chaperone is required for the conjugation reaction; however, it does not substitute for E3. Involvement of the chaperone in the proteolytic process requires complex formation with the substrate. Formation of this complex appears to be essential in the proteolytic process. In addition, the proper function of the chaperone in the proteolytic process requires the presence of K ؉ , which allows rapid cycles of dissociation and association of the complex. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site. In addition, it can also act directly on the ubiquitination machinery.Degradation of short-lived and key regulatory proteins via the ubiquitin-proteasome pathway plays important roles in basic cellular processes. Protein targets of the ubiquitin system include, among others, cyclins, cyclin-dependent kinases and their inhibitors, tumor suppressors, oncoproteins, and transcriptional activators and their inhibitors. Selection of proteins for degradation can be mediated via primary (constitutive) or secondary signals such as post-translational modifications or via association with ancillary proteins. These signals are recognized by specific ubiquitin-protein ligases (E3), 1 to which the substrate proteins bind prior to ubiquitination. Thus, the ligases play a key role in the ubiquitin proteolytic cascade, recognition and selection of proteins for conjugation and subsequent degradation. Following formation of the polyubiquitin adduct, the protein moiety is degraded by the 26 S proteasome complex, and free and reutilizable ubiquitin is released (reviewed in Refs. 1-4).Molecular chaperones comprise a set of universally conserved proteins that bind and stabilize conformers of other proteins. A regulated, ATP-dependent association-dissociation cyc...

show abstract

Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of .beta.,.gamma.-nonhydrolyzable ATP analogs

Cited by 42 publications

References 49 publications

Degradation of the Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) by the Ubiquitin-Proteasome Pathway

Degradation of the Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) by the Ubiquitin-Proteasome Pathway

CD28 Costimulation Mediates T Cell Expansion Via IL-2-Independent and IL-2-Dependent Regulation of Cell Cycle Progression

Ubiquitin-dependent Degradation of Certain Protein Substrates in Vitro Requires the Molecular Chaperone Hsc70

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