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:2.7.11.22 (cdc2)
8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex.
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PMID:Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase. 756 22

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.
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PMID:The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. 778 45

Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis.
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PMID:Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase. 912

Metaphase II arrest of Xenopus oocyte is characterized by the presence of M-phase-promoting factor (MPF) and of a microtubular spindle, both of which are stable in the presence of protein synthesis inhibitors. We studied in vivo this equilibrium state that is settled during meiotic maturation. At time of germinal vesicle breakdown (GVBD), cdc2 kinase and MAP kinase activities are stimulated. A component of the cyclin ubiquitin ligase, CDC27, is phosphorylated at the same time and remains phosphorylated until fertilization, indicating that an important component of the ligase complex is modified as early as GVBD. During a first period extending from GVBD until the cortical anchorage of the metaphase II spindle, homogeneous pools of cdc2 kinase and mitogen-activated protein (MAP) kinase activities are present in oocyte and are strictly dependent on protein turnover, since protein synthesis inhibition induces their total inactivation and drives oocytes into interphase. The metaphase II spindle, once anchored into the cortex, is no more sensitive to protein synthesis inhibition, likewise MAP kinase activity. During this cellular arrest, cdc2 kinase is divided into two distinctly regulated pools. The first one contains cyclin B that actively turns over and is subjected to a microtubular checkpoint. The second one is stable. Alteration of intracellular compartmentation of metaphase II oocytes either by gentle centrifugation or by cold shock inactivates MAP kinase and targets all cyclin B molecules for full destruction. We therefore suggest that MAP kinase participates to the cytostatic activity by preventing part of cyclin B molecules from entering the ubiquitination/degradation machinery which is still turned on in metaphase II oocytes.
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PMID:In vivo regulation of cytostatic activity in Xenopus metaphase II-arrested oocytes. 916 50

In S. cerevisiae, the G1/S transition requires Cdc4p, Cdc34p, Cdc53p, Skp1p, and the Cln/Cdc28p cyclin-dependent kinase (Cdk). These proteins are thought to promote the proteolytic inactivation of the S-phase Cdk inhibitor Sic1p. We show here that Cdc4p, Cdc53p, and Skp1p assemble into a ubiquitin ligase complex named SCFCdc4p. When mixed together, SCFCdc4p subunits, E1 enzyme, the E2 enzyme Cdc34p, and ubiquitin are sufficient to reconstitute ubiquitination of Cdk-phosphorylated Sic1p. Phosphorylated Sic1p substrate is specifically targeted for ubiquitination by binding to a Cdc4p/Skp1p subcomplex. Taken together, these data illuminate the molecular basis for the G1/S transition in budding yeast and suggest a general mechanism for phosphorylation-targeted ubiquitination in eukaryotes.
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PMID:A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. 934 31

The replication initiation protein Cdc6p forms a tight complex with Cdc28p, specifically with forms of the kinase that are competent to promote replication initiation. We now show that potential sites of Cdc28 phosphorylation in Cdc6p are required for the regulated destruction of Cdc6p that has been shown to occur during the Saccharomyces cerevisiae cell cycle. Analysis of Cdc6p phosphorylation site mutants and of the requirement for Cdc28p in an in vitro ubiquitination system suggests that targeting of Cdc6p for degradation is more complex than previously proposed. First, phosphorylation of N-terminal sites targets Cdc6p for polyubiquitination probably, as expected, through promoting interaction with Cdc4p, an F box protein involved in substrate recognition by the Skp1-Cdc53-F-box protein (SCF) ubiquitin ligase. However, in addition, mutation of a single, C-terminal site stabilizes Cdc6p in G2 phase cells without affecting substrate recognition by SCF in vitro, demonstrating a second and novel requirement for specific phosphorylation in degradation of Cdc6p. SCF-Cdc4p- and N-terminal phosphorylation site-dependent ubiquitination appears to be mediated preferentially by Clbp/Cdc28p complexes rather than by Clnp/Cdc28ps, suggesting a way in which phosphorylation of Cdc6p might control the timing of its degradation at then end of G1 phase of the cell cycle. The stable cdc6 mutants show no apparent replication defects in wild-type strains. However, stabilization through mutation of three N-terminal phosphorylation sites or of the single C-terminal phosphorylation site leads to dominant lethality when combined with certain mutations in the anaphase-promoting complex.
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PMID:Phosphorylation controls timing of Cdc6p destruction: A biochemical analysis. 1051 65

In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumour-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity remains unknown. Here, using an in vivo assay to measure protein stability in real time during the cell cycle, we show that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A resulted in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period.
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PMID:Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. 1054 10

Coordinated accumulation of cyclin D1 and D3 is observed in 15% of primary breast cancers and in the breast cancer cell line MCF-7 this simultaneous overexpression is due to a defect in their ubiquitin-mediated proteolysis. The F-box protein Skp2 is a component of an SCF ubiquitin ligase complex and can associate with cyclin D1 and the cdk inhibitor p21 (Zhong-Kang et al., 1998). We extend this observation and show that cyclin D3 can also associate with Skp2 suggesting that cyclins D1, D3 and p21 may share the same SCF complex. In agreement with this hypothesis we report here that in primary breast cancers and in MCF-7 cells where cyclins D1 and D3 are elevated the level of p21 is also elevated. Further, we demonstrate that the turnover of p21 protein is reduced in MCF-7 cells. We show that p21 is active as a cdk inhibitor in this cell line but that the presence of elevated levels of cyclin D3 titrates p21 away from cyclin D1-cdk4/6 complexes and cdk2 complexes resulting in increased kinase activities. Our results suggest that a defect in the SCF complex may occur in 15-20% of breast cancers and that the resulting coordinated elevation of cyclins D1 and D3 overcomes the inhibition of cell cycle progression by p21. We propose that in the context of cyclins D1 and D3 overexpression, p21 may promote cell cycle progression.
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PMID:Inhibitory effect of p21 in MCF-7 cells is overcome by its coordinated stabilization with D-type cyclins. 1059 47

The periodic expression of cell cycle proteins is important for the regulation of cell cycle progression. The amount of CDK inhibitor, p27(kip1), one such protein, seems to be regulated by the ubiquitin-proteasome system. The ubiquitin ligase (E3) toward p27(kip1) is thought to be SCF(skp2). The activity of SCF(skp2) was increased by the addition of Roc1 protein to the complex. Furthermore, the ubiquitination of p27(kip1) seemed to be dependent on the phosphorylation of T187 of p27(kip1) because the mutant T187A was not ubiquitinated at all in an in vitro ubiquitination system. Cullin-1, a component of SCF, is modified by ubiquitin-like protein Nedd8. The modification site of cullin-1 was shown to be K696 because the K696R mutant was not modified. When the effect of the Nedd8 modification on the SCF(skp2) activity toward p27(kip1) was investigated, the activity was markedly decreased by using the Nedd8-unmodified mutant cullin-1 (K696R), indicating that the modification may play an important role on the SCF(skp2) activity toward p27(kip1).
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PMID:Modification of cullin-1 by ubiquitin-like protein Nedd8 enhances the activity of SCF(skp2) toward p27(kip1). 1077 55

Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G(1), accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations in CDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G(1)-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1p(db-mut)) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCF(Met30) in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.
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PMID:Hsl1p, a Swe1p inhibitor, is degraded via the anaphase-promoting complex. 1084 88


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