Ligand-dependent kinase activity
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The synthesis, characterization and biological activity of the first zinc(II) complexes with potent inhibitors of cyclin-dependent kinases (CDKs) derived from 6-benzylaminopurine are described. Based on the results following from elemental analyses, infrared, NMR and ES+MS (electrospray mass spectra in the positive ion mode) spectroscopies, conductivity data, thermal analysis and X-ray structures, the tetrahedral Zn(II) complexes of the compositions [Zn(Olo)Cl(2)](n) (1), [Zn(iprOlo)Cl(2)](n) (2), [Zn(BohH(+))Cl(3)] x H(2)O (3) and [Zn(iprOloH(+))Cl(3)] x H(2)O (4) have been prepared, where Olo=2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine (Olomoucine), iprOlo=2-(2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (i-propyl-Olomoucine), Boh=2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpurine (Bohemine). The 1D-polymeric chain structure for [Zn(Olo)Cl(2)](n) (1) as well as the monomeric one for [Zn(BohH(+))Cl(3)] x H(2)O (3) and [Zn(iprOloH(+))Cl(3)] x H(2)O (4) have been revealed unambiguously by single crystal X-ray analyses. The 1D-polymeric chain of 1 consists of Zn(Olo)Cl(2) monomeric units in which the Zn(II) ion is coordinated by two chlorine atoms and one oxygen atom of the 2-hydroxyethylamino group of Olomoucine. The next monomeric unit is bonded to Zn(II) through the N7 atom of a purine ring. Thus, each of Zn(II) ions is tetrahedrally coordinated and a ZnCl(2)NO chromophore occurs in the complex 1. The complexes 3 and 4 are mononuclear species with a distorted tetrahedral arrangement of donor atoms around the Zn(II) ion with a ZnCl(3)N chromophore. The corresponding CDK inhibitor, i.e., both Boh and iprOlo, is coordinated to Zn(II) via the N7 atom of the purine ring in 3 and 4. The cytotoxicity of the zinc(II) complexes against human melanoma, sarcoma, leukaemia and carcinoma cell lines has been determined as well as the inhibition of the CDK2/cyclin E kinase. A relationship between the structure and biological activity of the complexes is also discussed.
- MeSH
- chloridy farmakologie chemie MeSH
- cyklin-dependentní kinasy antagonisté a inhibitory chemie MeSH
- diferenční termická analýza MeSH
- financování organizované MeSH
- hmotnostní spektrometrie MeSH
- inhibitory enzymů farmakologie chemická syntéza chemie MeSH
- kinetin farmakologie chemie MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- ligandy MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- organokovové sloučeniny farmakologie chemická syntéza chemie MeSH
- protinádorové látky farmakologie chemie MeSH
- puriny farmakologie chemie MeSH
- screeningové testy protinádorových léčiv MeSH
- sloučeniny zinku farmakologie chemie MeSH
- spektrofotometrie infračervená MeSH
- Check Tag
- lidé MeSH
14-3-3 proteiny jsou důležitými regulačními bílkovinami, jež se nacházejí ve všech eukaryotních organismech, v lidských buňkách tvoří rodinu 14-3-3 proteinů sedm isoforem. Funkce 14-3-3 proteinů spočívá v interakci s jejich vazebnými partnery, jichž zatím bylo identifikováno několik set. Tito partneři mají klíčové role v mnoha buněčných pochodech, jako je signalizace, regulace buněčného cyklu a dělení, apoptosa a další. PI4KB (fosfatidylinositol 4-kinasa B), nacházející se na cytosolové straně membrán převážně Golgiho aparátu, katalyzuje připojení fosfátové skupiny na čtvrtý uhlík inositolového kruhu fosfatidylinositolu. Aktivita PI4KB je závislá na fosforylaci serinu v pozici 294. Tato fosforylace nejen, že zvyšuje kinasovou aktivitu PI4KB, ale také je podmínkou pro vazbu 14-3-3 proteinů. Tato interakce zajišťuje ochranu PI4KB před defosforylací a tak zaručuje nepřetržitou syntézu fosfatidylinositol 4-fosfátu, významné signalizační molekuly a prekursoru pro další fosfátové deriváty fosfatidylinositolu.
The family of 14-3-3 proteins is one of great regulatory significance and can be found in all eukaryotic organisms and consists of seven isoforms in human cells. The function of 14-3-3 proteins rests in the interaction with their ligands of which several hundreds have been identified. The key role of these partners comes to pass in many cellular processes such as signalization, regulation of a cell cycle and division, apoptosis and others. Phosphatidylinositol 4-kinase B (PI4KB) is situated on the cytosolic side of mostly Golgi aparatus membranes and catalyzes attachment of a phosphate group to the fourth carbon of inositol group of phosphatidylinositol. The activity of PI4KB depends on the phosphorylation of Ser294. Not only this phosphorylation increases the kinase activity PI4KB, but is the condition for 14-3-3 proteins binding as well. This interaction provides the protection of PI4KB against dephosphorylation and this way it guarantees continual synthesis of phosphatidylinositol 4-phosphate (PI4P), a major signalization molecule and the precursor of other phosphate derivatives of phosphatidylinositol.
Cytochrome P450 1B1 (CYP1B1) is an enzyme that has a unique tumor-specific pattern of expression and is capable of bioactivating a wide range of carcinogenic compounds. We have reported previously that coordinated upregulation of CYP1B1 by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and the aryl hydrocarbon receptor ligands, may increase bioactivation of promutagens, such as benzo[a]pyrene (BaP) in epithelial cells. Here, we extend those studies by describing a novel mechanism participating in the regulation of CYP1B1 expression, which involves activation of the p38 mitogen-activated protein kinase (p38) and mitogen- and stress-activated protein kinase 1 (MSK1). Using inhibitors of p38 and MSKs, as well as mouse embryonic cells derived from p38α-deficient and MSK1/2 double knockout mice, we show here that TNF-α potentiates CYP1B1 upregulation via the p38/MSK1 kinase cascade. Effects of this inflammatory cytokine on CYP1B1 expression further involve the positive transcription elongation factor b (P-TEFb). The inhibition of the P-TEFb subunit, cyclin-dependent kinase 9 (CDK9), which phosphorylates RNA polymerase II (RNAPII), prevented the enhanced CYP1B1 induction by a combination of BaP and inflammatory cytokine. Furthermore, using chromatin immunoprecipitation assays, we found that cotreatment of epithelial cells with TNF-α and BaP resulted in enhanced recruitment of both CDK9 and RNAPII to the Cyp1b1 gene promoter. Overall, these results have implications concerning the contribution of inflammatory factors to carcinogenesis, since enhanced CYP1B1 induction during inflammation may alter metabolism of exogenous carcinogens, as well as endogenous CYP1B1 substrates playing role in tumor development.
- MeSH
- cyklin-dependentní kinasa 9 genetika MeSH
- cytochrom P450 CYP1B1 biosyntéza genetika MeSH
- cytokiny metabolismus MeSH
- karcinogeneze účinky léků genetika MeSH
- karcinogeny toxicita MeSH
- lidé MeSH
- mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory genetika metabolismus MeSH
- myši MeSH
- nádory chemicky indukované genetika patologie MeSH
- pozitivní transkripční elongační faktor b genetika MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- RNA-polymerasa II genetika MeSH
- signální transdukce účinky léků MeSH
- TNF-alfa metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.
- MeSH
- 2-naftylamin analogy a deriváty chemie MeSH
- adenosintrifosfát chemie metabolismus MeSH
- alosterická regulace MeSH
- alosterické místo MeSH
- AMP cyklický chemie metabolismus MeSH
- barvení a značení metody MeSH
- fluorescenční barviva chemie MeSH
- inhibitory proteinkinas chemie metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- lidé MeSH
- ligandy MeSH
- peptidy chemie metabolismus MeSH
- proteinkinasy závislé na cyklickém AMP chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The maturation of mammalian oocytes in vitro can be stimulated by gonadotropins (follicle-stimulating hormone, FSH) or their intrafollicular mediator, epidermal growth factor (EGF)-like peptide-amphiregulin (AREG). We have shown previously that in pig cumulus-oocyte complexes (COCs), FSH induces expression and the synthesis of AREG that binds to EGF receptor (EGFR) and activates the mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. However, in this study we found that FSH also caused a rapid activation of MAPK3/1 in the cumulus cells, which cannot be explained by the de novo synthesis of AREG. The rapid MAPK3/1 activation required EGFR tyrosine kinase (TK) activity, was sensitive to SRC proto-oncogene non-receptor tyrosine kinase (SRC)-family and protein kinase C (PKC) inhibitors, and was resistant to inhibitors of protein kinase A (PKA) and metalloproteinases. AREG also induced the rapid activation of MAPK3/1 in cumulus cells, but this activation was only dependent on the EGFR TK activity. We conclude that in cumulus cells, FSH induces a rapid activation of MAPK3/1 by the ligand-independent transactivation of EGFR, requiring SRC and PKC activities. This rapid activation of MAPK3/1 precedes the second mechanism participating in the generation and maintenance of active MAPK3/1-the ligand-dependent activation of EGFR depending on the synthesis of EGF-like peptides.
- MeSH
- aktivace transkripce MeSH
- amfiregulin metabolismus MeSH
- erbB receptory metabolismus MeSH
- extracelulárním signálem regulované MAP kinasy genetika MeSH
- folikuly stimulující hormon farmakologie MeSH
- IVM techniky MeSH
- kultivované buňky MeSH
- kumulární buňky cytologie účinky léků metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 1 genetika MeSH
- mitogenem aktivovaná proteinkinasa 3 genetika MeSH
- oocyty cytologie účinky léků metabolismus MeSH
- prasata MeSH
- signální transdukce účinky léků MeSH
- skupina kinas odvozených od src-genu metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The first Fe(III) complexes 1-6 with cyclin-dependent kinase (CDK) inhibitors of the type [Fe(L(n))Cl(3)].nH(2)O (n=0 for 1, 1 for 2, 2 for 3-6; L(1)-L(6)=C2- and phenyl-substituted CDK inhibitors derived from 6-benzylamino-9-isopropylpurine), have been synthesized and characterized by elemental analysis, IR, (57)Fe Mössbauer, (1)H and (13)C NMR, and ES+ mass spectroscopies, conductivity and magnetic susceptibility measurements, and thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The study revealed that the compounds are mononuclear, tetrahedral high-spin (S=5/2) Fe(III) complexes with an admixture of an S=3/2 spin state originating probably from five-coordinated Fe(III) ions either connecting with a bidentate coordination mode of the CDK inhibitor ligand or relating to the possibility that one crystal water molecule enters the coordination sphere of the central atom in a portion of molecules of the appropriate complex. Nearly spin-only value of the effective magnetic moment (5.82micro(eff)/micro(B)) was determined for compound 1 due to absence of crystal water molecule(s) in the structure of the complex. Based on NMR data and DFT calculations, we assume that the appropriate organic ligand is coordinated to the Fe(III) ion through the N7 atom of a purine moiety. The cytotoxicity of the complexes was tested in vitro against selected human cancer cell lines (G-361, HOS, K-562 and MCF-7) along with the ability to inhibit the CDK2/cyclinE kinase. The best cytotoxicity (IC(50): 4-23muM) and inhibition activity (IC(50): 0.02-0.09microM) results have been achieved in the case of complexes 2-4, and complexes 3, 4 and 6, respectively. In addition, the X-ray structure of 2-chloro-6-benzylamino-9-isopropylpurine, i.e. a precursor for the preparation of L(1), L(4) and L(5), is also described.
- MeSH
- elektrochemické techniky metody MeSH
- inhibiční proteiny cyklin-dependentních kinas chemie metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- protinádorové látky chemická syntéza chemie farmakologie MeSH
- puriny chemie MeSH
- screeningové testy protinádorových léčiv MeSH
- spektrální analýza metody MeSH
- železo chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We studied hsBAFF activity in in vitro mouse splenic B cells. hsBAFF effects on intracellular free Ca2+ concentration ([Ca2+]i) were assayed, using a laser scanning confocal microscope with fluorescent probe, Fluo-3/AM. We showed that treatment of B cells with 0.5-5 µg/ml hsBAFF resulted in significantly higher [Ca2+]i levels in a dose-dependent fashion at 12 and 24 h, respectively (p<0.05 or p<0.01 vs. control). Furthermore, we noticed that 2.5 µg/ml hsBAFF-treated cells were significantly resistant to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca2+-ATPase inhibitor (p<0.05 hsBAFF plus Tg group vs. Tg group). Thus hsBAFF may promote B cell survival by direct upregulation of [Ca2+]i physiological homeostasis contributing to prevention of [Ca2+]i dysfunction. Using immunocytochemistry and Western blot analysis, we found that the activation of ERK1/2 due to hsBAFF was triggered by a [Ca2+]i-dependent pathway, leading to elevation of B cell proliferation. This is supported by the findings that intracellular Ca2+ chelator BAPTA/AM attenuated phosphorylated ERK1/2 expression and cell proliferation in hsBAFF-stimulated B cells. hsBAFF-stimulated B cell proliferation was obviously reduced by mitogen extracellular kinase 1/2 (MEK1/2, upstream of ERK1/2) inhibitor U0126. Taken together, the main finding of this study is that hsBAFF elicits higher but homeostatic [Ca2+]i levels, which regulates ERK1/2 activity and cell proliferation in in vitro B cells
- MeSH
- B-lymfocyty enzymologie MeSH
- butadieny farmakologie MeSH
- časové faktory MeSH
- chelátory farmakologie MeSH
- EGTA analogy a deriváty farmakologie MeSH
- faktor aktivující B-buňky metabolismus MeSH
- financování organizované MeSH
- fosforylace MeSH
- homeostáza MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- mitogenem aktivovaná proteinkinasa 1 metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 3 metabolismus MeSH
- myši inbrední ICR MeSH
- myši MeSH
- nitrily farmakologie MeSH
- proliferace buněk MeSH
- sarkoplazmatická Ca2+-ATPáza antagonisté a inhibitory metabolismus MeSH
- thapsigargin farmakologie MeSH
- vápník metabolismus MeSH
- viabilita buněk MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
The heart is the first organ required to function during embryonic development and is absolutely necessary for embryo survival. Cardiac activity is dependent on both the sinoatrial node (SAN), which is the pacemaker of heart's electrical activity, and the cardiac conduction system which transduces the electrical signal though the heart tissue, leading to heart muscle contractions. Defects in the development of cardiac electrical function may lead to severe heart disorders. The Erbb2 (Epidermal Growth Factor Receptor 2) gene encodes a member of the EGF receptor family of receptor tyrosine kinases. The Erbb2 receptor lacks ligand-binding activity but forms heterodimers with other EGF receptors, stabilising their ligand binding and enhancing kinase-mediated activation of downstream signalling pathways. Erbb2 is absolutely necessary in normal embryonic development and homozygous mouse knock-out Erbb2 embryos die at embryonic day (E)10.5 due to severe cardiac defects. We have isolated a mouse line, l11Jus8, from a random chemical mutagenesis screen, which carries a hypomorphic missense mutation in the Erbb2 gene. Homozygous mutant embryos exhibit embryonic lethality by E12.5-13. The l11Jus8 mutants display cardiac haemorrhage and a failure of atrial function due to defects in atrial electrical signal propagation, leading to an atrial-specific conduction block, which does not affect ventricular conduction. The l11Jus8 mutant phenotype is distinct from those reported for Erbb2 knockout mouse mutants. Thus, the l11Jus8 mouse reveals a novel function of Erbb2 during atrial conduction system development, which when disrupted causes death at mid-gestation.
- MeSH
- akční potenciály MeSH
- missense mutace MeSH
- myši inbrední C57BL MeSH
- myši kmene 129 MeSH
- myši transgenní MeSH
- převodní systém srdeční embryologie patofyziologie MeSH
- receptor erbB-2 genetika metabolismus MeSH
- srdce - funkce síní MeSH
- srdeční síně embryologie metabolismus patofyziologie MeSH
- vrozené srdeční vady genetika patofyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
