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:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

20S proteasomes are large (700 kDa) proteinase complexes which form the central catalytic core of larger complexes (26S proteasomes or PA28-20S complexes) formed by association with regulatory particles. These larger complexes are involved in diverse regulatory processes in the cell including cyclin breakdown, proteolytic control of transcription factors and other short-lived regulatory proteins, and antigen presentation. In order to carry out these diverse functions the proteasome complexes must be held under tight regulatory control. The early recognition of potential phosphorylation sites in a number of core and regulatory subunits suggested that some control of the complexes activities may be via phosphorylation. We have investigated the role of phosphorylation in determining proteasome localization, activities and association with regulatory complexes.
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PMID:Phosphorylation of proteasomes in mammalian cells. 1036 40

The CDC25 dual specificity phosphatase is a universal cell cycle regulator. The evolutionary conservation of this enzyme from yeast to man bears witness to its major role in the control of cyclin-dependent kinases (CDK) activity that are central regulators of the cell cycle machinery. CDC25 phosphatase both dephosphorylates and activates CDKs. Three human CDC25s have been identified. CDC25A is involved in the control of G1/S, and CDC25C at G2/M throught the activation of CDK 1-cyclin B. The exact function of CDC25B however remains elusive. We have found that CDC25B is degraded by the proteasome pathway in vitro and in vivo. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex, but not by CDK1-cyclin B. Together with the observations of others made in yeast and mammals, our results suggest that CDC25B might act as a 'mitotic starter' triggering the activation of an auto-amplification loop before being degraded.
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PMID:Proteasome-dependent degradation of human CDC25B phosphatase. 1036 47

The CDK inhibitor, p21WAF1/Cip1 blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.
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PMID:Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway. 1043 39

The ubiquitin proteasome pathway regulates the expression of major cellular regulatory proteins. The ubiquitin proteasome system has been demonstrated to be involved in the expression of the cyclin kinase inhibitor, p21. Ubiquitinated p21 is degraded immediately by 26S proteasome, therefore, the detection of p21 is difficult. We report here an improvement for the detection of ubiquitinated p21 using a proteasome inhibitor, clasto-lactacystin beta-lactone. A p21-enriched cell lysate is obtained by pretreating the cells with deferoxamine to induce p21 mRNA expression followed by treatment with 1x10(-6) M beta-lactone. The concentration of p21 from the cell lysate was performed using an anti-p21 antibody crosslinked to protein G Sepharose. Ubiquitinated p21 was detected on Western blots of the concentrated sample using an anti-ubiquitin antibody. This detection system will be used for further analysis of the regulation of p21 ubiquitination.
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PMID:Direct proteasome inhibition by clasto-lactacystin beta-lactone permits the detection of ubiquitinated p21(waf1) in ML-1 cells. 1044 2

Retinoids have demonstrated activity in the chemoprevention of aerodigestive tract cancer. Potentially contributing to their lung cancer chemopreventive effects, retinoids inhibit the growth of human bronchial epithelial (HBE) cells. We observed previously that all-trans retinoic acid (t-RA) arrests the growth of HBE cells in the G0 phase of the cell cycle through activation of retinoic acid receptor-dependent pathways, which enhances the association of E2F-4 with retinoblastoma protein family members, converting E2F into a transcriptional suppressor. In this study, we examined the mechanism by which t-RA blocks cell cycle progression in HBE cells and the possibility that this signaling event is blocked in non-small cell lung cancer (NSCLC) cells that are refractory to the growth inhibitory effects of t-RA. t-RA suppressed the expression and activity of cyclin D1, cyclin E, and cyclin-dependent kinases (CDK)-2 and CDK-4, increased expression of the CDK inhibitor p27, and shifted the retinoblastoma protein to a hypophosphorylated form. Posttranslational mechanisms contributed to the changes in CDK-2, CDK-4, and p27 levels, which, in the case of CDK-4, involved the ubiquitin-proteasome pathway. In contrast, despite retinoic acid receptor transcriptional activation, these signaling events did not occur in a NSCLC cell line that is refractory to growth inhibition by t-RA. These findings provide the first evidence that t-RA activates degradation of CDK-4 through the ubiquitin-proteasome pathway, a novel mechanism by which t-RA causes HBE cells to exit the cell cycle, and blockade of these signaling events may contribute to the development of retinoid resistance in NSCLC cells.
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PMID:Posttranslational mechanisms contribute to the suppression of specific cyclin:CDK complexes by all-trans retinoic acid in human bronchial epithelial cells. 1044 3

By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G(1)/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G(1)-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G(1) arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.
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PMID:Functional characterization of rpn3 uncovers a distinct 19S proteasomal subunit requirement for ubiquitin-dependent proteolysis of cell cycle regulatory proteins in budding yeast. 1049 Jun 25

Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. We pursued the first of these pathways by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin-protein ligases. We found that Cul-3 was bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice caused increased accumulation of cyclin E protein, and had cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there was a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance.
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PMID:Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells. 1050 95

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.
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PMID:SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis. 1058 39

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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PMID:CD28 costimulation mediates T cell expansion via IL-2-independent and IL-2-dependent regulation of cell cycle progression. 1060 5

Both chromosome segregation and the final exit from mitosis require a ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome. This multiprotein complex ubiquitinates various substrates, such as the anaphase inhibitor Pds1 and mitotic cyclins, and thus targets them for proteolysis by the 26S proteasome. The ubiquitination by APC is dependent on the presence of a destruction-box sequence in the N-terminus of target proteins. Recent reports have strongly suggested that Cdc20, a WD40 repeat-containing protein required for nuclear division in the budding yeast Saccharomyces cerevisiae, is essential for the APC-mediated proteolysis. To understand the function of CDC20, we have studied its regulation in some detail. The expression of the CDC20 gene is cell-cycle regulated such that it is transcribed only during late S phase and mitosis. Although the protein is unstable to some extent through out the cell cycle, its degradation is particularly enhanced in G1. Cdc20 contains a destruction box sequence which, when mutated or deleted, stabilizes it considerably in G1. Surprisingly, we find that while the inactivation of APC subunits Cdc16, Cdc23 or Cdc27 results in stabilization of the mitotic cyclin Clb2 in G1, the proteolytic destruction of Cdc20 remains largely unaffected. This suggests the existence of proteolytic mechanisms in G1 that can degrade destruction-box containing proteins, such as Cdc20, in an APC-independent manner.
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PMID:Cdc20 protein contains a destruction-box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase-promoting complex. 1063 13


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