Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In eukaryotic cells, many short-lived proteins are conjugated with Lys 48-linked ubiquitin chains and degraded by the proteasome. Ubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). Most ubiquitin ligases use either a HECT (homologous to E6-associated protein C terminus) or a RING (really interesting new gene) domain to catalyse polyubiquitination, but the mechanism of E3 catalysis is poorly defined. Here we dissect this process using mouse Ube2g2 (E2; identical at the amino acid level to human Ube2g2) and human gp78 (E3), an endoplasmic reticulum (ER)-associated conjugating system essential for the degradation of misfolded ER proteins. We demonstrate by expressing recombinant proteins in Escherichia coli that Ube2g2/gp78-mediated polyubiquitination involves preassembly of Lys 48-linked ubiquitin chains at the catalytic cysteine of Ube2g2. The growth of Ube2g2-anchored ubiquitin chains seems to be mediated by an aminolysis-based transfer reaction between two Ube2g2 molecules that each carries a ubiquitin moiety in its active site. Intriguingly, polyubiquitination of a substrate can be achieved by transferring preassembled ubiquitin chains from Ube2g2 to a lysine residue in a substrate.
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PMID:A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate. 1731 Jan 45

Cyclin A is targeted for mitotic destruction by the anaphase promoting complex/cyclosome (APC/C) and degradation proceeds even when proteolysis of other APC/C substrates are blocked by the spindle assembly checkpoint. Instead of a simple destruction box, a complex N-terminal destruction signal has been implicated in Cyclin A. We show here that Drosophila Cyclin A destruction employs both N- and C-terminal residues, which emphasize that a synergistic action by different parts of the protein facilitates recognition and degradation. The first KEN box, first D-box and an aspartic acid at position 70 are required at the N-terminus and they make additive contributions when the spindle checkpoint is active. From the C-terminal region, the cyclin box contributes. Single point mutations in these four elements abolish mitotic destruction. Additionally, eight lysines in the neighborhood of the N-terminal signals, which could serve as potential ubiquitin acceptor sites, are preferentially used for proteolysis. Mutations in these lysines and the N-terminal signals cause mitotic stability. However, mutating the lysines alone, only delays mitotic progression. Thus, presumably, lysines elsewhere in the protein are used when the preferred ones are absent and this requires the N-terminal signals. Furthermore, our results suggest that some function of the cyclin box other than Cdk1 binding promotes spindle checkpoint-independent recognition of Cyclin A by the APC/C.
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PMID:Cyclin A degradation employs preferentially used lysines and a cyclin box function other than Cdk1 binding. 1731 14

M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca(++)/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.
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PMID:Role for non-proteolytic control of M-phase-promoting factor activity at M-phase exit. 1732 11

We reported here an efficient and generally applicable genomic analysis that uses transcriptional profiling to identify candidate substrates of regulatory enzymes, such as kinases and ubiquitin ligases. We applied this strategy to the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls sister chromatid separation and exit from mitosis. We found that a microtubule-associated protein, CKAP2, is a substrate of APC/C and demonstrated that ubiquitination and degradation of CKAP2 in vitro require a KEN-box and is mediated by Cdh1, an activator of APC/C. We showed that the levels of CKAP2 fluctuated across the cell cycle in culture cells, high in mitosis and low during mitotic exit. Overexpression of Cdh1 reduced the levels of CKAP2 in a KEN-box-dependent manner, while knockdown of Cdh1 increased the half-life of CKAP2. CKAP2 associated with centrosomal microtubules in late G(2), but only after the separation of the duplicated centrosomes. During mitosis, CKAP2 associated with spindle poles and with spindle microtubules from prophase through anaphase and dis-appeared from microtubules during cytokinesis. The function of CKAP2 during mitosis does not seem essential, as efficient knockdown of CKAP2 neither altered the cell cycle distribution of the cells, nor generated observable mitotic defects. On the other hand, ectopic expression of either the wild-type or a non-degradable CKAP2 led to a mitotic arrest with monopolar spindles containing highly bundled microtubules. We concluded that CKAP2 is a physiological substrate of APC/C during mitotic exit and that a tight regulation of the CKAP2 protein level is critical for the normal mitotic progression.
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PMID:CKAP2 is a spindle-associated protein degraded by APC/C-Cdh1 during mitotic exit. 1737 72

The fidelity of cell division is dependent on the accumulation and ordered destruction of critical protein regulators. By triggering the appropriately timed, ubiquitin-dependent proteolysis of the mitotic regulatory proteins securin, cyclin B, aurora A kinase, and polo-like kinase 1, the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays an essential role in maintaining genomic stability. Misexpression of these APC/C substrates, individually, has been implicated in genomic instability and cancer. However, no comprehensive survey of the extent of their misregulation in tumors has been performed. Here, we analyzed more than 1600 benign and malignant tumors by immunohistochemical staining of tissue microarrays and found frequent overexpression of securin, polo-like kinase 1, aurora A, and Skp2 in malignant tumors. Positive and negative APC/C regulators, Cdh1 and Emi1, respectively, were also more strongly expressed in malignant versus benign tumors. Clustering and statistical analysis supports the finding that malignant tumors generally show broad misregulation of mitotic APC/C substrates not seen in benign tumors, suggesting that a "mitotic profile" in tumors may result from misregulation of the APC/C destruction pathway. This profile of misregulated mitotic APC/C substrates and regulators in malignant tumors suggests that analysis of this pathway may be diagnostically useful and represent a potentially important therapeutic target.
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PMID:Oncogenic regulators and substrates of the anaphase promoting complex/cyclosome are frequently overexpressed in malignant tumors. 1745 82

Pathways of the molecular pathogenesis of colorectal carcinoma have been extensively studied and molecular lesions during the development of the disease have been revealed. High up in the list of colorectal cancer lesions are APC (adenomatous polyposis coli), K-ras, Smad4 (or DPC4-deleted in pancreatic cancer 4) and p53 genes. All these molecules are part of important pathways for the regulation of cell proliferation and apoptosis and as a result perturbation of these processes lead to carcinogenesis. The ubiquitin-proteasome system (UPS) is comprised of a multi-unit cellular protease system that regulates several dozens of cell proteins after their ligation with the protein ubiquitin. Given that among these proteins are regulators of the cell cycle, apoptosis, angiogenesis, adhesion and cell signalling, this system plays a significant role in cell fate and carcinogenesis. UPS inhibition has been found to be a pre-requisite for apoptosis and is already clinically exploited with the proteasome inhibitor bortezomib in multiple myeloma. Cyclooxygenase-2 (Cox-2) is the inducible form of the enzyme that metabolizes the lipid arachidonic acid to prostaglandin H2, the first step of prostaglandins production. This enzyme is up-regulated in colorectal cancer and in several other cancers. Inhibition of Cox-2 by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) has been found to inhibit proliferation of colorectal cancer cells and in epidemiologic studies has been shown to reduce colon polyp formation in genetically predisposed populations and in the general population. NSAIDs have also Cox-independent anti-proliferative effects. Targeted therapies, the result of increasingly understanding carcinogenesis in the molecular level, have entered the field of anti-neoplastic treatment and are used by themselves and in combination with chemotherapy drugs. Combinations of targeted drugs have started also to be investigated. This article reviews the molecular pathogenesis of colorectal cancer, the roles of UPS and Cox-2 in it and puts forward a rational for their combined inhibition in colorectal cancer treatment.
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PMID:Pathogenesis of colorectal carcinoma and therapeutic implications: the roles of the ubiquitin-proteasome system and Cox-2. 1748 76

Mounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G(1) arrest associated with elevated p27(Kip1) and p21 (WAF1) proteins. The adhesion-mediated p27(Kip1) and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCF(Skp2) ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27(Kip1), whereas inhibition of Skp2 by siRNA increased p27(Kip1) and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27(Kip1) degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27(Kip1) signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.
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PMID:Cell adhesion induces p27Kip1-associated cell-cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas. 1750 56

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage.
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PMID:Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage. 1756 51

Ubiquitin-dependent proteolysis plays an important role in regulating fundamental biological functions, including cell division and cellular differentiation. Previous studies implicate the ubiquitin-proteasome system (UPS) in myogenic differentiation through regulating cell cycle progression and modulating myogenic factors such as MyoD and Myf5. Certain ubiquitin protein ligases, including the SCF complex and APC, have been suggested to govern terminal muscle differentiation. However, the underlying mechanism of regulation of both the cell cycle and myogenic factors by the UPS during this process remains unclear. We have dissected the role of the UPS in myogenic differentiation using an in vitro muscle differentiation system based on C2C12 cells. We demonstrate that Cdh1-APC regulates two critical proteins, Skp2 and Myf5, for proteolysis during muscle differentiation. The targeting of Skp2 by Cdh1-APC for destruction results in elevation of p21 and p27, which are crucial for coordinating cellular division and differentiation. Degradation of Myf5 by Cdh1-APC facilitates myogenic fusion. Knockdown of Cdh1 by siRNA significantly attenuates muscle differentiation. Taken together, Cdh1-APC is an important ubiquitin E3 ligase that modulates muscle differentiation through coordinating cell cycle progression and initiating the myogenic differentiation program.
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PMID:The dual effects of Cdh1/APC in myogenesis. 1760 83

RNF8 is a ubiquitin ligase with a FHA domain near its N terminus, and a RING-finger domain at its C terminus, through which it recruits several ubiquitin-conjugating enzymes. In metazoans, only the mitotic checkpoint regulator CHFR shares this domain architecture. Here we show that RNF8 is a nuclear protein that follows a cell-cycle-dependent turnover, reaching its highest levels in mitosis, followed by a strong decline in late mitotic stages. Overexpression of RNF8 caused a delay in cytokinesis and the frequent appearance of aberrant mitotic figures. These effects were dependent on the ubiquitin ligase activity of RNF8, since they were significantly attenuated when a RING-finger mutant, inactive as an E3, was overexpressed. Depletion of RNF8 also caused a delay in the exit from the mitotic arrest induced by nocodazole, associated with a reduced turnover of the APC/C substrate cyclin B1. These observations suggest that RNF8 regulates the rate of exit from mitosis and cytokinesis.
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PMID:Regulation of mitotic exit by the RNF8 ubiquitin ligase. 1772 60


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