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)

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly homologous in sequence or structure to other dual-specificity phosphatases, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. Like other dual-specificity phosphatases, Cdc25 contains an active site cysteine whose pK(a) of 5.9 can be measured in pH-dependent kinetics using both small molecule and protein substrates such as Cdk2-pTpY/CycA. We have previously shown that the catalytic acid expected in phosphatases of this family and apparent in kinetics with the natural protein substrate does not appear to lie within the known structure of Cdc25 [Chen, W., et al. (2000) Biochemistry 39, 10781]. Here we provide experimental evidence for a novel mechanism wherein Cdc25 uses as its substrate a monoprotonated phosphate in contrast to the more typical bisanionic phosphate. Our pH-dependent studies, including one-turnover kinetics, solvent kinetic isotope effects, equilibrium perturbation, substrate depletion, and viscosity measurements, show that the monoprotonated phosphate of the protein substrate Cdk2-pTpY/CycA provides the critical proton to the leaving group. Additionally, we provide evidence that Glu474 on the Cdc25 enzyme serves an important role as a base in the transfer of the proton from the phosphate to the leaving group. Because of its greater intrinsic reactivity, the use of a monoprotonated phosphate as a phosphatase substrate is a chemically attractive solution and suggests the possibility of designing inhibitors specific for the Cdc25 dual-specificity phosphatase, an important anticancer target.
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PMID:Catalytic mechanism of Cdc25. 1246 61

Oxidative stress has been demonstrated to produce modifications in several intracellular proteins that lead to alterations in their activities. Alzheimer's disease is related to an increase of oxidative stress markers, which may be an early event in the progression of the disease and neurofibrillary tangles formation. Abnormal phosphorylation of tau has been implicated in the etiopathogenesis of Alzheimer's disease. By using phospho-specific antibodies, we analyzed the changes in tau phosphorylation patterns after treatment of rat hippocampal and SHSY5Y human neuroblastoma cells with H2O2. We found that tau isoforms were hypophosphorylated at the Tau1 epitope after 2 h in the presence of H2O2. The decrease in the phosphorylation levels of tau protein were prevented by pretreatment with N-acetyl-L-cysteine. These changes were shown to depend on the activity of the cdk5/p35 complex, since a 3-fold increase in substrate phosphorylation and a 2-fold increase for the complex association were observed. Also, a decrease in the amount of inhibitor-2 bound to phosphatase PP1 was found in SHSY5Y cells under oxidative stress conditions. This decrease of inhibitor-2 bound to PP1 is due to an increased phosphorylation of the inhibitor-2 protein, thus leading to increased PP1 activity. Therefore, we propose that oxidative stress-induced activation of cdk5 leads to inhibitor-2 phosphorylation, relieving its inhibitory effect on PP1.
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PMID:Oxidative stress promotes tau dephosphorylation in neuronal cells: the roles of cdk5 and PP1. 1513 75

Anticancer effects of the dietary isothiocyanate sulforaphane were investigated in the human pancreatic cancer cell lines MIA PaCa-2 and PANC-1. Sulforaphane-treated cells accumulated in metaphase as determined by flow cytometry [4C DNA content, cyclin A(-), cyclin B1(+), and phospho-histone H3 (Ser(10))(+)]. In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-8, loss of mitochondrial membrane potential, and loss of plasma membrane integrity. The initial detection of caspase-3 cleavage occurring in G(2)-M arrest was independent of a change in phospho-cdc2 (Tyr(15)) protein; consequently, sulforaphane treatment combined with UCN-01 had no significant impact on cellular toxicity. Incubations at higher sulforaphane doses (>10 micromol/L) resulted in cleavage of caspase-3 in the G(1) subpopulation, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated. Cellular toxicity in MIA PaCa-2, and to a greater extent in PANC-1, was positively correlated with a decrease in cellular glutathione levels, whereas sustained increases in glutathione observed in MIA PaCa-2 cells or the simultaneous incubation with N-acetyl-L-cysteine in PANC-1 cells were associated with resistance to sulforaphane-induced apoptosis. Daily sulforaphane i.p. injections (375 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with PANC-1 s.c. tumors resulted in a decrease of mean tumor volume by 40% compared with vehicle-treated controls. Our findings suggest that, in addition to the known effects on cancer prevention, sulforaphane may have activity in established pancreatic cancer.
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PMID:The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice. 1548 91

Sustained activation of ERK 1/2 by a low dose (15 mg/kg ip) of S-1,2-dichlorovinyl-l-cysteine (DCVC) 72 h before administration of a lethal dose of DCVC (75 mg/kg ip) enhances renal cell division and protects mice against acute renal failure (ARF) and death (autoprotection). The objective of this study was to determine correlation among extent of S-phase DNA synthesis, activation of transcription factors, expression of G(1)/S cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors downstream of ERK 1/2 following DCVC-induced ARF in autoprotection. Administration of the lethal dose alone caused a general downregulation or an unsustainable increase, in transcriptional and posttranscriptional events thereby preventing G(1)-S transition of renal cell cycle. Phosphorylation of IkappaBalpha was inhibited resulting in limited nuclear translocation of NF-kappaB. However, cyclin D1 expression was high probably due to transcriptional cooperation of AP-1. Cyclin D1/cyclin-dependent kinase 4 (cdk4)-cdk6 system-mediated phosphorylation of retinoblastoma protein was downregulated due to overexpression of p16 at 24 h after exposure to the lethal dose alone. Inhibition of S-phase stimulation was confirmed by proliferating cell nuclear antigen assay (PCNA). This inhibitory response was prevented if the lethal dose was administered 72 h after the low priming dose of DCVC due to promitogenic effect of the low dose. NF-kappaB-DNA binding is not limited if mice were pretreated with the priming dose. Cyclin D1/cdk4-cdk6 expression stimulated by the priming dose of DCVC was unaltered even after the lethal dose in the autoprotected group, explaining higher phosphorylated-pRB and S-phase stimulation found in this group. These results were corroborated with PCNA immunohistochemistry. These findings suggest that the priming dose relieves the block on compensatory tissue repair by upregulation of promitogenic mechanisms, normally blocked by the high dose when administered without the prior priming dose.
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PMID:Molecular mechanisms of enhanced renal cell division in protection against S-1,2-dichlorovinyl-L-cysteine-induced acute renal failure and death. 1574 5

Recent findings suggested that the role of cysteine proteases would not be limited to protein degradation in lysosomes but would also play regulatory functions in more specific cell mechanisms. We analyzed here the role of these enzymes in the control of cell cycle during embryogenesis. The addition of the potent cysteine protease inhibitor E64d to newly fertilized sea urchin eggs disrupted cell cycle progression, affecting nuclear as well as cytoplasmic characteristic events. Monitoring BrdU incorporation in E64d treated eggs demonstrated that DNA replication is severely disturbed. Moreover, this drug treatment inhibited male histones degradation, a step that is necessary for sperm chromatin remodeling and precedes the initiation of DNA replication in control eggs. This inhibition likely explains the DNA replication disturbance and suggests that S phase initiation requires cysteine protease activity. In turn, activation of the DNA replication checkpoint could be responsible for the consecutive block of nuclear envelope breakdown (NEB). However, in sea urchin early embryos this checkpoint doesn't control the mitotic cytoplasmic events that are not tightly coupled with NEB. Thus the fact that microtubule spindle is not assembled and cyclin B-cdk1 not activated under E64d treatment more likely rely on a distinct mechanism. Immunofluorescence experiments indicated that centrosome organization was deficient in absence of cysteine protease activity. This potentially accounts for mitotic spindle disruption and for cyclin B mis-localization in E64d treated eggs. We conclude that cysteine proteases are essential to trigger S phase and to promote M phase entry in newly fertilized sea urchin eggs.
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PMID:Inhibition of cysteine protease activity disturbs DNA replication and prevents mitosis in the early mitotic cell cycles of sea urchin embryos. 1579 98

Heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro (GI50 < 500 nmol/L). In vivo growth inhibition of renal, colon, and breast xenografts has been observed. Profound G2-M cell cycle block accompanied down-regulation of cdk1 gene transcription was corroborated by decreased CDK1 protein expression following treatment of HCT 116 cells with growth inhibitory concentrations of 1 or 2. The chemical structure of the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) suggested that these novel agents would readily react with nucleophiles in a double Michael (beta-carbon) addition. Indeed, COMPARE analysis within the National Cancer Institute database revealed a number of chemically related quinone derivatives that could potentially react with sulfur nucleophiles in a similar manner and suggested that thioredoxin/thioredoxin reductase signal transduction could be a putative target. Molecular modeling predicted covalent irreversible binding between quinol analogues and cysteine residues 32 and 35 of thioredoxin, thereby inhibiting enzyme activity. Binding has been confirmed, via mass spectrometry, between reduced human thioredoxin and 1. Microarray analyses of untreated HCT 116 cells and those exposed to either 1 (1 micromol/L) or 2 (500 nmol/L and 1 micromol/L) determined that of > or =10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC50 < 6 micromol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin.
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PMID:Elucidation of thioredoxin as a molecular target for antitumor quinols. 1586 91

The cytoskeleton is critical to neuronal functioning and survival. Cytoskeletal alterations are involved in several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We studied the possible pathways involved in colchicine-induced apoptosis in cerebellar granule neurons (CGNs). Although colchicine evoked an increase in caspase-3, caspase-6 and caspase-9 activation, selective caspase inhibitors did not attenuate apoptosis. Inhibitors of other cysteine proteases such as PD150606 (a calpain-specific inhibitor), Z-Phe-Ala fluoromethyl ketone (a cathepsins-inhibitors) and N(alpha)-p-tosyl-l-lysine chloromethyl ketone (serine-proteases inhibitor) also had no effect on cell death/apoptosis induced by colchicine. However, BAPTA-AM 10 microM (intracellular calcium chelator) prevented apoptosis mediated by cytoskeletal alteration. These data indicate that calcium modulates colchicine-induced apoptosis in CGNs. PARP-1 inhibitors did not prevent apoptosis mediated by colchicine. Finally, colchicine-induced apoptosis in CGNs was attenuated by kenpaullone, a cdk5 inhibitor. Kenpaullone and indirubin also prevented cdk5/p25 activation mediated by colchicine. These findings indicate that cytoskeletal alteration can compromise cdk5 activation, regulating p25 formation and suggest that cdk5 inhibitors attenuate apoptosis mediated by cytoskeletal alteration. The present data indicate the potential therapeutic value of drugs that prevent the formation of p25 for the treatment of neurodegenerative disorders.
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PMID:Evaluation of the neuronal apoptotic pathways involved in cytoskeletal disruption-induced apoptosis. 1595 Sep 51

Protein kinase C (PKC) represents a family of serin/threonine kinases, playing a central role in the regulation of cell growth, differentiation and transformation. These enzymes differ in their primary structure, biochemical properties, tissue distribution and subcellular localization. The specific cellular functions of PKC isoforms are largely controlled by their localization. PKCeta, a member of the novel subfamily, is expressed predominantly in epithelial tissues. However, not much is known with respect to its mechanism of activation and regulation. Our recent studies suggest its role in cell cycle control. Here we show that PKCeta is localized at the Golgi apparatus, ER and the nuclear envelope. Furthermore, using GFP-fusion proteins of the different functional domains of PKCeta we deciphered the specific structural domains of the protein responsible for its apparent localization. We show that the cysteine-rich repeat C1b is responsible for its Golgi localization, while for its presence at the ER/nuclear envelope the pseudosubstrate containing fragment coupled to the C1 domain is required. In response to short-term activation by PMA we show translocation of PKCeta to the plasma membrane and the nuclear envelope. We demonstrate that the C1b is sufficient for its translocation to the plasma membrane. Interestingly, accumulation of PKCeta at the nuclear envelope also occurred in response to serum-starvation. It should be noted that interaction of PKCeta with the cyclin E/Cdk2 complex at the perinuclear region was recently reported by us in response to serum-starvation. Thus, our studies demonstrate translocation of PKCeta to the nuclear envelope, and suggest that the spatial regulation of PKCeta could be important for its cellular functions including effects on cell cycle control and involvement in tumor promotion.
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PMID:PKCeta is localized in the Golgi, ER and nuclear envelope and translocates to the nuclear envelope upon PMA activation and serum-starvation: C1b domain and the pseudosubstrate containing fragment target PKCeta to the Golgi and the nuclear envelope. 1624 15

Roscovitine is a specific inhibitor of cyclin-dependent kinases (cdks) cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35. The studies on the enzyme inhibitory properties and cellular effects of roscovitine revealed that it arrests cells in G(2)/M and G(1)/S phase, inhibits the proliferation of mammalian cells and induces cell death. However, the characteristics of cell death and exact mechanism by which this cdk inhibitor kills transformed cells are unknown. We previously investigated that the roscovitine induces apoptotic death of mitotic PC12 cells. The present study was to identify whether the roscovitine-induced death is related with the specific elements of caspases in pathway of apoptosis. The morphological data of caspase-3 immunofluorocytochemistry double staining with hoechst 33342 indicated that apoptotic nuclei were identified as nuclei with chromatin condensation and nuclear fragmentation, and that caspase-3 active p17 subunit co-existed in PC12 cells treated with roscovitine 50 micromol/L for 4 h. The number of the caspase-3 positive cells increased significantly to about 42%, as compared with the normal control (P<0.001). The data of MTT assay showed that the number of viable cells treated by roscovitine (50 micromol/L) alone for 12 h was 29.03%, of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 mumol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 micromol/L) increased viable PC12 cells to 45.16%, (Z-DEVD-FMK) and 58.06%, (Z-VAD-FMK), respectively, in the presence of roscovitine. Non-erythroid a-spectrin is a cytoskeleted protein that is a substrate of caspase-3 cysteine proteases. To confirm the activity of caspase-3 that produced in roscovitine (50 micromol/L for 12 h)-induced PC12 cell death, activated caspase-3 specific 120 kDa spectrin breakdown products (SBDP) were detected by Western bloting using the mouse anti-non-erythroid a-spectrin monoclonal antibody. The mean relative density of bands corresponding to caspase-3 specific SBDP levels were significantly increased in the cytosolic fractions treated with roscovitine, as compared to the normal control (P<0.001). These results indicate that caspase signals, especially caspase-3 signal are necessary for the progression of proliferating PC12 cell apoptotic death evoked by roscovintine.
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PMID:[Caspase-3 plays a required role in PC12 cell apoptotic death induced by roscovitine]. 1634 2

Calpains are a large family of Ca2+-dependent cysteine proteases that are ubiquitously distributed across most cell types and vertebrate species. Calpains play a role in cell differentiation, apoptosis, cytoskeletal remodeling, signal transduction and the cell cycle. The cell cycle proteins cyclin D1 and p21(KIP1), for example, have been shown to be affected by calpains. However, the rules that govern calpain cleavage specificity are poorly understood. We report here studies on the pattern of mu-calpain proteolysis of the p19(INK4d) protein, a cyclin-dependent kinase 4/6 inhibitor that negatively regulates the mammalian cell cycle. Our data show new characteristics of calpain action: mu-calpain cleaves p19(INK4d) immediately after the first and second ankyrin repeats that are structurally less stable compared to the other repeats. This is in contrast to features observed so far in the specificity of calpains for their substrates. These results imply that calpain may be involved in the cell cycle by regulating the cell cycle regulatory protein turnover through CDK inhibitors and cyclins.
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PMID:Identification of calpain cleavage sites in the G1 cyclin-dependent kinase inhibitor p19(INK4d). 1654 56


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