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Protein Kinase Inhibitors (protein + kinase_inhibitor)
Selected AbstractsEpidermal Growth Factor Induces Oxidative Neuronal Injury in Cortical CultureJOURNAL OF NEUROCHEMISTRY, Issue 1 2000Yoo Kyung Cha Abstract : Recently, we have demonstrated that certain neurotrophic factors can induce oxidative neuronal necrosis by acting at the cognate tyrosine kinase-linked receptors. Epidermal growth factor (EGF) has neurotrophic effects via the tyrosine kinase-linked EGF receptor (EGFR), but its neurotoxic potential has not been studied. Here, we examined this possibility in mouse cortical culture. Exposure of cortical cultures to 1-100 ng/ml EGF induced gradually developing neuronal death, which was complete in 48-72 h ; no injury to astrocytes was noted. Electron microscopic findings of EGF-induced neuronal death were consistent with necrosis ; severe mitochondrial swelling and disruption of cytoplasmic membrane occurred, whereas nuclei appeared relatively intact. The EGF-induced neuronal death was accompanied by increased free radical generation and blocked by the anti-oxidant Trolox. Suggesting mediation by the EGFR, an EGFR tyrosine kinase-specific inhibitor, C56, attenuated EGF-induced neuronal death. In addition, inhibitors of extracellular signal-regulated protein kinase 1/2 (Erk-1/2) (PD98056), protein kinase A (H89), and protein kinase C (GF109203X) blocked EGF-induced neuronal death. A p38 mitogen-activated protein kinase inhibitor (SB203580) or glutamate antagonists (MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione) showed no protective effect. The present results suggest that prolonged activation of the EGFR may trigger oxidative neuronal injury in central neurons. [source] A CALCIUM-DEPENDENT PROTEIN KINASE FUNCTIONS IN WOUND HEALING IN VENTRICARIA VENTRICOSA (CHLOROPHYTA)JOURNAL OF PHYCOLOGY, Issue 6 2000Koh-ichi Sugiyama The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa ( J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca2+. Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti- Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca2+, and a 40-kDa Ca2+ -independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca2+ -dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48 / 80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca2+ receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound. [source] Effect of inhibitors of mitogen-activated protein kinase kinase on ,1B -adrenoceptor phosphorylationAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 1-2 2009R. Alcántara-Hernández Summary 1,Mitogen-activated protein kinases mediate hormone/neurotransmitter action on proliferation and differentiation and participate in receptor regulation. The effect of inhibitors of mitogen-activated kinase kinase (MEK) on ,1B -adrenoceptor phosphorylation state and function was studied using different cell lines. It was observed that at nanomolar concentrations the MEK inhibitors, PD98059 (2,-amino-3,-methoxyflavone) and UO126 [1,4-(diamino-2,3-dicyano/1,4-bis-(2-aminophenylthio)-butadiene], increased ,1B -adrenoceptor phosphorylation and diminished the functional response of this receptor to noradrenaline. These agents did not alter the action of lysophosphatidic acid. 2,Staurosporine (IC50 , 0.8 nm) (a general protein kinase inhibitor) and bis-indolyl-maleimide I (IC50 , 200 nm) (a selective protein kinase C inhibitor) inhibited PD98059-induced ,1B -adrenoceptor phosphorylation. In contrast, neither wortmannin (phosphoinositide 3-kinase inhibitor) nor genistein (protein tyrosine kinase inhibitor) had any effect. The data suggest the possibility that MEK might exert control on the activity of the enzymes that regulate receptor phosphorylation, such as G-protein-coupled receptor kinases, protein kinase C or serine/threonine protein phosphatases. 3,Coimmunoprecipitation studies showed a constant association of total extracellular signal-regulated kinase 2 (ERK2) with ,1B -adrenoceptors. Association of phospho-ERK 1/2 to ,1B -adrenoceptors increased not only in response to agonist but also in response to agents that increase ,1B -adrenoceptor and ERK1/2 phosphorylation [such as endothelin-1, phorbol 12-myristate-13-acetate (PMA) and epidermal growth factor (EGF)]; not surprisingly, PD98059 decreased this effect. 4,Our data show that blockade of MEK activity results in increased ,1B -adrenoceptor phosphorylation, diminished adrenoceptor function and perturbation of receptor,ERK1/2 interaction. [source] E230Q mutation of the catalytic subunit of cAMP-dependent protein kinase affects local structure and the binding of peptide inhibitorBIOPOLYMERS, Issue 6 2006Man-Un Ung Abstract The active site of the mammalian cAMP-dependent protein kinase catalytic subunit (C-subunit) has a cluster of nonconserved acidic residues,Glu127, Glu170, Glu203, Glu230, and Asp241,that are crucial for substrate recognition and binding. Studies have shown that the Glu230 to Gln mutant (E230Q) of the enzyme has physical properties similar to the wild-type enzyme and has decreased affinity for a short peptide substrate, Kemptide. However, recent experiments intended to crystallize ternary complex of the E230Q mutant with MgATP and protein kinase inhibitor (PKI) could only obtain crystals of the apo-enzyme of E230Q mutant. To deduce the possible mechanism that prevented ternary complex formation, we used the relaxed-complex method (Lin, J.-H., et al. J Am Chem Soc 2002, 24, 5632,5633) to study PKI binding to the E230Q mutant C-subunit. In the E230Q mutant, we observed local structural changes of the peptide binding site that correlated closely to the reduced PKI affinity. The structural changes occurred in the F-to-G helix loop and appeared to hinder PKI binding. Reduced electrostatic potential repulsion among Asp241 from the helix loop section and the other acidic residues in the peptide binding site appear to be responsible for the structural change. © 2005 Wiley Periodicals, Inc. Biopolymers 81: 428,439, 2006 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Enhanced sensitivity to cis -diamminedichloro-platinum(II) of a human carcinoma cell line with mutated p53 gene by cyclin-dependent kinase inhibitor p21WAF1 expressionCANCER SCIENCE, Issue 3 2003Mamoru Satou p53-mediated induction of p21WAF1, a cyclin-dependent protein kinase inhibitor, is known to protect cancer cells from the cytotoxic effects of anti-cancer drugs or ,-irradiation. Since the p53 gene is frequently inactivated in cancer cells, we examined whether p21WAF1 expression may alter the sensitivity of cancer cells with mutated p53 gene to anti-cancer drugs. Cells of a colon cancer cell line DLD-1 were transfected with p21WAF1 expression vector controlled by a tetracycline-repressable promoter and transfec-tants were cloned (Dp21,1). p21WAF1 expression induced by removal of tetracycline from culture media repressed cell proliferation and resulted in altered cell shape, suggesting induction of differentiation. Dp21,1 cells with p21WAF1 expression were more sensitive to cis -diamminedichloroplatinum(II) (CDDP) (IC50 value, 10 ,M) than those without p21WAF1 expression (IC50, 22 ,M). Sensitivity to doxorubicin was not different between Dp21,1 cells with and without p21WAF1 expression. DNA ladder formation was observed in Dp21,1 cells treated with CDDP, indicating that the enhanced sensitivity to CDDP involves apoptosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosolic protein revealed that subunit protein bands with Mr 55 kDa and 44 kDa were markedly increased in cells with p21WAF1 expression. By im-munoblotting, these proteins were identified as c-Jun N-terminal kinase (JNK) 2 and p38 mitogen-activated protein kinase (MAPK) 8, respectively, both of which are believed to be involved in apoptosis induction by CDDP. These results suggest that p21WAF1 may enhance the sensitivity of colon cancer cells with mutated p53 gene to CDDP, possibly through the JNK and p38 MAPK pathways. (Cancer Sci 2003; 94: 286,291) [source] Off-Target Decoding of a Multitarget Kinase Inhibitor by Chemical ProteomicsCHEMBIOCHEM, Issue 7 2009Enrico Missner Abstract Unbiased: Chemical proteomics was used to profile compound interactions in an unbiased fashion. We present here the application of different compound-immobilization routes for decoding nonprotein kinase off-targets of the multitarget kinase inhibitor C1, which interacts with distinct compound moieties. Since the approval of the first selective tyrosine kinase inhibitor, imatinib, various drugs have been developed to target protein kinases. However, due to a high degree of structural conservation of the ATP binding site, off-target effects have been reported for several drugs. Here, we report on off-target decoding for a multitarget protein kinase inhibitor by chemical proteomics, by focusing on interactions with nonprotein kinases. We tested two different routes for the immobilization of the inhibitor on a carrier matrix, and thus identified off-targets that interact with distinct compound moieties. Besides several of the kinases known to bind to the compound, the pyridoxal kinase (PDXK), which has been described to interact with the CDK inhibitor (R)-roscovitine, was captured. The PDXK,inhibitor interaction was shown to occur at the substrate binding site rather than at the ATP binding site. In addition, carbonic anhydrase 2 (CA2) binding was demonstrated, and the determination of the IC50 revealed an enzyme inhibition in the submicromolar range. The data demonstrate that different compound immobilization routes for chemical proteomics approaches are a valuable method to improve the knowledge about the off-target profile of a compound. [source] Bacterial protein kinase inhibitorsDRUG DEVELOPMENT RESEARCH, Issue 3 2010Michio Kurosu Abstract Protein kinases have become the second most important group of drug targets for the pharmaceutical industry next to G-protein-coupled receptors. Thus, over the past decade, a significant number of small molecules have been generated for protein kinase drug optimization programs. The vast majority of kinase inhibitors target the ATP binding site of the enzyme; however, the poor protein kinase selectivity of ATP-competitive protein kinase inhibitors (PKIs) limits their use for treating chronic diseases. In contrast, for inhibitors of bacterial signal transduction systems targeting bacterial kinase(s), there are no such selectivity requirements as long as the inhibitor does not act on any human kinases at the effective concentrations for killing bacteria in vivo. Protein phosphorylation in bacteria is performed by two-component signal transduction systems (2CSTSs) and eukaryotic-like serine/threonine kinases or bacterial tyrosine kinases. Recently, a large number of studies of protein kinases essential for sustaining bacterial growth and kinases required for virulence have been reported. Thus, bacterial protein kinases offer considerable potential as new drug targets. To identify bacterial PKIs, large chemical libraries of ATP-competitive inhibitors developed for eukaryotic protein kinases are an invaluable asset. This manuscript reviews progress on the development of prokaryotic protein kinase inhibitors. Drug Dev Res 2010. © 2010 Wiley-Liss, Inc. [source] Brief exposure to NMDA produces long-term protection of cerebellar granule cells from apoptosisEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2005Xavier Xifro Abstract Cerebellar granule cells (CGCs) require excitatory inputs to survive during their postnatal migration from the external to the internal granule cell layers. The lack of innervation of mossy fibres induces CGC death by apoptosis. In vitro, CGCs die by apoptosis in the presence of physiological concentrations of KCl (5 mm or K5) but they survive in the presence of depolarizing concentrations of KCl (25 mm or K25) or N -methyl- d -aspartate (NMDA) by a mechanism dependent on calcium influx. The addition of NMDA or K25, for only 24 h, to immature CGCs cultures [2 days in vitro (DIV)] was able to produce a remarkable and long-term protection until 8 DIV. Moreover, our data show that NMDA and K25-mediated long-lasting protection was related to an inhibition of caspase-3 activity. By using different protein kinase inhibitors, we have shown that the inhibition of caspase-3 activation by NMDA was dependent on the activation of tyrosine kinases and phosphatidylinositol 3-kinase (PI3-kinase). Moreover, an impairment in NMDA-mediated neuroprotection and caspase-3 inhibition was observed when the action of brain-derived neurotrophic factor (BDNF) was blocked. By contrast, K25-mediated neuroprotection was BDNF-independent and was mediated by a mitogen-activated protein kinase- and PI3-kinase-dependent inhibition of caspase-3. [source] Transcriptional regulation of tumor necrosis factor-, in keratinocytes mediated by interleukin-1, and tumor necrosis factor-,EXPERIMENTAL DERMATOLOGY, Issue 6 2002S. Lisby Abstract: Irritant contact dermatitis (ICD) is an inflammatory skin reaction in which cytokines are thought to play a crucial role. In particular, tumor necrosis factor-, (TNF-,) has been implicated in the mechanism of this reaction. We report that interleukin-1, (IL-1,) that has been reported up-regulated in many inflammatory skin conditions is capable of increasing TNF-, mRNA and protein expression in murine keratinocytes. Furthermore, we show that TNF-, is capable of up-regulating itself in keratinocytes most likely in an autocrine manner. The signalling mechanisms involved in both IL-1,- and TNF-,-mediated regulation of TNF-, are critically dependent upon protein kinase C (PKC), as demonstrated by blocking studies using protein kinase inhibitors. Furthermore, the increase in TNF-, mRNA expression seen after stimulation with rTNF-, and rIL-1, involved increased transcription of TNF-, mRNA. This was demonstrated in a chloramphenicol acetyltransferase (CAT) assay using a CAT-construct containing the full-length TNF-, promoter. These observations support the notion of keratinocytes functioning as an amplifier of pro-inflammatory cytokine generation in the epidermis during ICD and other inflammatory skin conditions. [source] Targeted inhibition of the EGFR pathways enhances Zn-BC-AM PDT-induced apoptosis in well-differentiated nasopharyngeal carcinoma cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Ho-Kee Koon Abstract Epidermal growth factor receptor (EGFR), a receptor often expressed in nasopharyngeal carcinoma (NPC) cells, is one of the recently identified molecular targets in cancer treatment. In the present study, the effects of combined treatment of Zn-BC-AM PDT with an EGFR inhibitor AG1478 were investigated. Well-differentiated NPC HK-1 cells were subjected to PDT with 1,µM of Zn-BC-AM and were irradiated at a light dose of 1,J/cm2 in the presence or absence of EGFR inhibitor AG1478. Specific protein kinase inhibitors of downstream EGFR targets were also used in the investigation. EGFR, Akt, and ERK were found constitutively activated in HK-1 cells and the activities could be inhibited by the EGFR inhibitor AG1478. A sub-lethal concentration of AG1478 was found to further enhance the irreversible cell damage induced by Zn-BC-AM PDT in HK-1 cells. Pre-incubation of the cells with specific inhibitors of EGFR (AG1478), PI3k/Akt (LY294002), or MEK/ERK (PD98059) before light irradiation were found to enhance Zn-BC-AM PDT-induced formation of apoptotic cells. The efficacy of Zn-BC-AM PDT can be increased through the inhibition of EGFR/PI3K/Akt and EGFR/MEK/ERK signaling pathways in NPC cells. Combination therapy with Zn-BC-AM PDT and EGFR inhibitors may further be developed for the treatment of advanced NPC. J. Cell. Biochem. 108: 1356,1363, 2009. © 2009 Wiley-Liss, Inc. [source] Overexpression of RGPR-p117 enhances regucalcin gene promoter activity in cloned normal rat kidney proximal tubular epithelial cells: Involvement of TTGGC motifJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Natsumi Sawada Abstract A novel protein RGPR-p117 was discovered as regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. RGPR-p117 is localized in the nucleus of kidney cells, and overexpression of RGPR-p117 can modulate regucalcin protein and its mRNA expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. This study was undertaken to determine whether overexpression of RGPR-p117 enhances the regucalcin promoter activity using the ,710/+18 LUC construct (wild-type) or ,710/+18 LUC construct (mutant) with deletion of ,523/,435 including TTGGC motif. NRK52E cells (wild-type) or stable HA-RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Wild-type cells or transfectants were transfected with the ,710/+18 LUC construct vector or the ,710/+18 LUC construct with deletion of ,523/,435. Wild-type cells or transfectants with subconfluency were cultured for 48 h in a DMEM medium containing either vehicle, BS (5%), or parathyroid hormone (1,34) (PTH; 10,7 M). Luciferase activity in wild-type cells was significantly increased with culture of BS or PTH. This increase was significantly blocked in the presence of various protein kinase inhibitors (staurosporine and PD 98059). Luciferase activity in transfectants was significantly increased as compared with that of wild-type cells in the absence of BS or PTH. The increase in luciferase activity in transfectants was completely decreased in mutant with deletion of ,523/,435 sequence of regucalcin promoter. This was also seen using the ,710/+18 LUC construct with deletion of ,523/,503 sequence containing TTGGC motif. The increase in luciferase activity in transfectants was not significantly enhanced with culture of BS (5%), PTH (10,7 M), Bay K 8644 (10,6 M), phorbol 12-myristate 13-acetate (PMA; 10,6 M), or N6, 2,-dibutyryl cyclic adenosine 3,, 5,-monophosphate (DcAMP; 10,4 M). The increase in luciferase activity in transfectants was completely inhibited with culture of dibucaine (10,6 M), staurosporine (10,9 M), PD 98059 (10,8 M), wortmannin (10,8 M), genistein (10,6 M), vanadate (10,6 M), or okadaic acid (10,6 M) which are inhibitors of various kinases and protein phosphatases. This study demonstrates that RGPR-p117 can enhance the regucalcin promoter activity which is related to the NF-1 consensus sequences including TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation and dephosphorylation in NRK52E cells. J. Cell. Biochem. 99: 589,597, 2006. © 2006 Wiley-Liss, Inc. [source] Can MM-PBSA calculations predict the specificities of protein kinase inhibitors?JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 16 2006Christopher S. Page Abstract An application of the Molecular mechanics Poisson,Boltzmann surface area (MM-PBSA) protocol to the prediction of protein kinase inhibitor selectivity is presented. Six different inhibitors are placed in equivalent orientations in each of six different receptors. Fully solvated molecular dynamics is then run for 1 ns on each of the 36 complexes, and the resulting trajectories scored, using the implicit solvent model. The results show some correlation with experimentally-determined specificities; anomalies may be attributed to a variety of causes, including difficulties in quantifying induced fit penalties and variabilities in normal modes calculations. Decomposing interaction energies on a per-residue basis yields more useful insights into the natures of the binding modes and suggests that the real value of such calculations lies in understanding interactions rather than outright prediction. © 2006 Wiley Periodicals, Inc. J Comput Chem, 2007 [source] Tumor necrosis factor-alpha (TNF-,) regulates Toll-like receptor 2 (TLR2) expression in microgliaJOURNAL OF NEUROCHEMISTRY, Issue 4 2007Mohsin Md. Abstract Microglia represent one effector arm of CNS innate immunity as evident by their role in pathogen recognition. We previously reported that exposure of microglia to Staphylococcus aureus (S. aureus), a prevalent CNS pathogen, led to elevated Toll-like receptor 2 (TLR2) expression, a pattern recognition receptor capable of recognizing conserved structural motifs associated with gram-positive bacteria such as S. aureus. In this study, we demonstrate that the proinflammatory cytokine tumor necrosis factor-, (TNF-,) enhances TLR2 expression in microglia, whereas interleukin-1, has no significant effect. To determine the downstream signaling events responsible for elevated microglial TLR2 expression in response to TNF-,, a series of signal transduction inhibitors were employed. Treatment with caffeic acid phenethyl ester, an inhibitor of redox-mediated nuclear factor-kappa B activation, significantly attenuated TNF-,-induced TLR2 expression. Similar results were observed with the IKK-2 and I,B-, inhibitors SC-514 and BAY 11-7082, respectively. In contrast, no significant alterations in TLR2 expression were observed with protein kinase C or p38 mitogen-activated protein kinase inhibitors. A definitive role for TNF-, was demonstrated by the inability of S. aureus to augment TLR2 expression in microglia isolated from TNF-, knockout mice. In addition, TLR2 expression was significantly attenuated in brain abscesses of TNF-, knockout mice. Collectively, these results indicate that in response to S. aureus, TNF-, acts in an autocrine/paracrine manner to enhance TLR2 expression in microglia and that this effect is mediated, in part, by activation of the nuclear factor-kappa B pathway. [source] Phosphatidylethanol Mediates its Effects on the Vascular Endothelial Growth Factor via HDL Receptor in Endothelial CellsALCOHOLISM, Issue 2 2009Marja Katriina Liisanantti Background:, Previous epidemiological studies have shown that light to moderate alcohol consumption has protective effects against coronary heart disease but the mechanisms of the beneficial effect of alcohol are not known. Ethanol may increase high density lipoprotein (HDL) cholesterol concentration, augment the reverse cholesterol transport, or regulate growth factors or adhesion molecules. To study whether qualitative changes in HDL phospholipids mediate part of the beneficial effects of alcohol on atherosclerosis by HDL receptor, we investigated whether phosphatidylethanol (PEth) in HDL particles affects the secretion of vascular endothelial growth factor (VEGF) by a human scavenger receptor CD36 and LIMPII analog-I (CLA-1)-mediated pathway. Methods:, Human EA.hy 926 endothelial cells were incubated in the presence of native HDL or PEth-HDL. VEGF concentration and CLA-1 protein expression were measured. Human CLA-1 receptor-mediated mechanisms in endothelial cells were studied using CLA-1 blocking antibody and protein kinase inhibitors. Results:, Phosphatidylethanol-containing HDL particles caused a 6-fold increase in the expression of CLA-1 in endothelial cells compared with the effect of native HDL. That emergent effect was mediated mainly through protein kinase C and p44/42 mitogen-activated protein kinase pathways. PEth increased the secretion of VEGF and that increase could be abolished by a CLA-1 blocking antibody. Conclusions:, High density lipoprotein particles containing PEth bind to CLA-1 receptor and thereby increase the secretion of VEGF from endothelial cells. Ethanol-induced protective effects against coronary heart disease may be explained, at least partly, by the effects of PEth-modified HDL particles on VEGF via CLA-1-mediated mechanisms in endothelial cells. [source] Abscisic acid activates acid invertases in developing grape berryPHYSIOLOGIA PLANTARUM, Issue 2 2005Qiu-Hong Pan Acid invertases play a key role in sugar metabolism, and the plant hormone abscisic acid (ABA) enhances sugar accumulation in crop sink organs, but information about the relationship between ABA and acid invertases has been limited. The present experiments were done with both in vivo pre-incubation of the grape (Vitis vinifera × V. labrusca L.) berry tissues in ABA-containing medium and in vivo infiltration of ABA into the intact berries. The results show that ABA activates both the soluble and cell wall-bound acid invertases during fruit development by enhancing their activities and amounts as assessed by immunoblotting or enzyme-linked immunosorbent assay. This activation was pH, time course and ABA dose dependent. The serine/threonine protein kinase inhibitors K252a, staurosporine and H7 and acid phosphatase increased the activation of ABA-induced acid invertase, but the tyrosine protein kinase inhibitor quercetin strongly suppressed the ABA-induced effects, suggesting that a complex reversible protein phosphorylation is involved in the ABA-induced activation of acid invertases. The effects of the protein kinase inhibitors were dependent on the in vivo state of the tissues but independent of the expression of acid invertases. Two ABA analogues, (,)-ABA and trans-ABA, had no effect on acid invertases, showing that the ABA-induced activation of acid invertases is specific to the physiologically active form of ABA. These data suggest that ABA may be involved in fruit development by activating acid invertases. [source] Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheatTHE PLANT JOURNAL, Issue 3 2009Ayalew Ligaba Summary In this study we examined the role of protein phosphorylation/dephosphorylation in the transport properties of the wheat (Triticum aestivum) root malate efflux transporter underlying Al resistance, TaALMT1. Pre-incubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurosporine) strongly inhibited both basal and Al3+ -enhanced TaALMT1-mediated inward currents (malate efflux). Pre-incubation with phosphatase inhibitors (okadaic acid and cyclosporine A) resulted in a modest inhibition of the TaALMT1-mediated currents. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), enhanced TaALMT1-mediated inward currents. Since these observations suggest that TaALMT1 transport activity is regulated by PKC-mediated phosphorylation, we proceeded to modify candidate amino acids in the TaALMT1 protein in an effort to identify structural motifs underlying the process regulating phosphorylation. The transport properties of eight single point mutations (S56A, S183A, S324A, S337A, S351-352A, S384A, T323A and Y184F) generated in amino acid residues predicted to be phosphorylation sites and examined electrophysiologically. The basic transport properties of mutants S56A, S183A, S324A, S337A, S351-352A, T323A and Y184F were not altered relative to the wild-type TaALMT1. Likewise the sensitivity of these mutants to staurosporine resembled that observed for the wild-type transporter. However, the mutation S384A was noticeable, as in oocytes expressing this mutant protein TaALMT1-mediated basal and Al-enhanced currents were significantly inhibited, and the currents were insensitive to staurosporine or PMA. These findings indicate that S384 is an essential residue regulating TaALMT1 activity via direct protein phosphorylation, which precedes Al3+ enhancement of transport activity. [source] Simultaneous signalling through c-mpl, c-kit and CXCR4 enhances the proliferation and differentiation of human megakaryocyte progenitors: possible roles of the PI3-K, PKC and MAPK pathwaysBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2001Hitoshi Minamiguchi We assessed the effect of signalling through CXCR4 on the proliferation and differentiation of human megakaryocytic progenitor cells (CFU-Meg) in the presence or absence of stem cell factor (SCF) and/or thrombopoietin (TPO), using peripheral blood-derived CD34+IL-6R, cells as a target. TPO alone induced a significant number of CFU-Meg colonies. Although stromal cell-derived factor-1 (SDF-1) or SCF alone did not support CFU-Meg colony formation, these factors had a synergistic effect on CFU-Meg colony formation in the presence of TPO. The combination of SDF-1, SCF and TPO induced twice as many CFU-Meg colonies as TPO alone. To investigate the mechanism of this synergistic action, we examined the effects of various protein kinase inhibitors on CFU-Meg colony formation. LY294002 and GF109203X (inhibitors of PI3-K and PKC respectively) completely or partially inhibited this synergistic action. In contrast, a MEK inhibitor (PD98059) did not inhibit CFU-Meg colony formation. It significantly increased the higher ploidy classes (16N to 64N) of megakaryocytes supported by TPO, TPO + SCF, TPO + SDF-1, and TPO + SCF + SDF-1, whereas it abolished the effect of SDF-1 on the increase of higher ploidy classes of megakaryocytes supported by TPO. These results suggest that MAPK may negatively or positively regulate the nuclear maturation of megakaryocytes, known as endomitosis. In the presence of PD98059, proplatelet formation (PPF) was significantly augmented, suggesting that the MAPK pathway may also inhibit the initiation of PPF. In conclusion, simultaneous activation of three signals through c-mpl, c-kit and CXCR4 can induce the in vitro proliferation and differentiation of CFU-Meg, and SDF-1 is a potentiator of human megakaryocytopoiesis. [source] KMUP-1 activates BKCa channels in basilar artery myocytes via cyclic nucleotide-dependent protein kinasesBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2005Bin-Nan Wu This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (KDR)- or large-conductance Ca2+ -activated potassium (BKCa) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+ - and Ca2+ channel activities. KMUP-1 (1 ,M) had no effect on the KDR current but dramatically enhanced BKCa channel activity. This increased BKCa current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BKCa current. BKCa current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 ,M), an adenylate cyclase inhibitor (SQ 22536 10 ,M), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 ,M and Rp-cAMP, 100 ,M), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM). Voltage-dependent L-type Ca2+ current was significantly suppressed by KMUP-1 (1 ,M), and nearly abolished by a calcium channel blocker (nifedipine, 1 ,M). In conclusion, KMUP-1 stimulates BKCa currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries. British Journal of Pharmacology (2005) 146, 862,871. doi:10.1038/sj.bjp.0706387 [source] Activation of NF-,B and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascadesCELLULAR MICROBIOLOGY, Issue 12 2003Guntram A. Grassl Summary Yersinia enterocolitica triggers activation of the nuclear factor (NF)-,B and production of the proinflammatory chemokine interleukin (IL)-8 in intestinal epithelial cells. This activation is due to adhesion of the bacteria via their outer membrane protein invasin to the host cells. Using Clostridium difficile toxins that specifically inactivate small GTPases, and transfection of inhibitory proteins of the Rho-GTPases, we demonstrate that Rac1, but not Cdc42 or Rho, is required for activation of NF-,B by invasin. Invasin activated the mitogen activated protein kinases (MAPK) p38 and c-Jun N-terminal protein kinase (JNK) but not extracellular signal regulated kinase (ERK). The functional relevance of these pathways for invasin-mediated IL-8 expression was assessed by protein kinase inhibitors and dominant-negative kinase mutants. While NF-,B and JNK contribute to IL-8 transcription, p38 MAPK also acts through stabilization of IL-8 mRNA, as confirmed by quantitative RT-PCR and electrophoretic mobility shift assays. Transfection experiments with I-,B kinase (IKK)1 and IKK2 mutants indicate that the release of NF-,B from its cytoplasmic inhibitor I-,B and its translocation into the nucleus is mediated by these kinases. Our data identify Rac1 as a key intermediate in invasin-triggered IL-8 synthesis and demonstrate that maximum IL-8 induction involves several MAP kinase cascades. [source] Pityriarubins, Novel Highly Selective Inhibitors of Respiratory Burst from Cultures of the Yeast Malassezia furfur: Comparison with the Bisindolylmaleimide Arcyriarubin ACHEMBIOCHEM, Issue 12 2005Hans-Joachim Krämer Dr. Abstract Pityriasis versicolor is the most common skin mycosis in humans worldwide. Yeasts of the genus Malassezia, particularly M. furfur, a saprophyte occurring widely on human skin, are generally regarded as the causative agents. M. furfur is able to convert tryptophan into a variety of indole alkaloids, some of them showing biological properties that correlate well with certain clinical features of pityriasis versicolor. This suggests a possible role for these compounds in the pathophysiology of the disease. We here report that the novel pityriarubins A, B and C, isolated from cultures of the yeast, inhibit respiratory burst in human neutrophils, activated by various agents, in a highly selective, unexpected manner. The release of 5-lipoxygenase products after challenge of neutrophils with the calcium ionophore A23187 is also inhibited in a dose-dependent manner. These activities reflect the close structural relationship of pityriarubins to bisindolylmaleimides, which have recently gained great interest as protein kinase inhibitors. [source] Functional Classification of Protein Kinase Binding Sites Using CavbaseCHEMMEDCHEM, Issue 10 2007Daniel Kuhn Dr. Abstract Increasingly, drug-discovery processes focus on complete gene families. Tools for analyzing similarities and differences across protein families are important for the understanding of key functional features of proteins. Herein we present a method for classifying protein families on the basis of the properties of their active sites. We have developed Cavbase, a method for describing and comparing protein binding pockets, and show its application to the functional classification of the binding pockets of the protein family of protein kinases. A diverse set of kinase cavities is mutually compared and analyzed in terms of recurring functional recognition patterns in the active sites. We are able to propose a relevant classification based on the binding motifs in the active sites. The obtained classification provides a novel perspective on functional properties across protein space. The classification of the MAP and the c-Abl kinases is analyzed in detail, showing a clear separation of the respective kinase subfamilies. Remarkable cross-relations among protein kinases are detected, in contrast to sequence-based classifications, which are not able to detect these relations. Furthermore, our classification is able to highlight features important in the optimization of protein kinase inhibitors. Using small-molecule inhibition data we could rationalize cross-reactivities between unrelated kinases which become apparent in the structural comparison of their binding sites. This procedure helps in the identification of other possible kinase targets that behave similarly in "binding pocket space" to the kinase under consideration. [source] Are MAP Kinases Drug Targets?CHEMMEDCHEM, Issue 8 2007but Difficult Ones Abstract Pharmaceutical companies are facing an increasing interest in new target identification and validation. In particular, extensive efforts are being made in the field of protein kinase inhibitors research and development, and the past ten years of effort in this field have altered our perception of the potential of kinases as drug targets. Therefore, in the drug discovery process, the selection of relevant, susceptible protein kinase targets combined with searches for leads and candidates have become a crucial approach. The success of recent launches of protein kinase inhibitors (Gleevec, Imatinib, Sutent, Iressa, Nexavar, Sprycel) gave another push to this field. Numerous other kinase inhibitors are currently undergoing clinical trials or clinical development. Some questions are nevertheless unanswered, mostly related to the great number of known kinases in the human genome, to their similarity with each other, to the existence of functionally redundant kinases for specific pathways, and also because the connection between particular pathways and diseases is not always clear. The review is leading the reader through a panoramic view of protein kinase inhibition with a major focus on MAPK, successful examples and clinical candidates. [source] | ||||||||||||||||||||||||||||