Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinases are emerging as one of the most intensely studied classes of enzymes as their central roles in physiologically and clinically important cellular signaling events become more clearly understood. We report here the development of a real-time, label-free method to study protein kinase inhibitor binding kinetics using surface plasmon resonance-based biomolecular interaction analysis (Biacore). Utilizing p38alpha mitogen-activated protein kinase as a model system, we studied the binding properties of two known small molecule p38alpha inhibitors (SB-203580 and SKF-86002). Direct coupling of p38alpha to the biosensor surface in the presence of a reversible structure-stabilizing ligand (SB-203580) consistently produced greater than 90% active protein on the biosensor surface. The dissociation and kinetic constants derived using this Biacore method are in excellent agreement with values determined by other methods. Additionally, we extend the method to study the thermodynamics of small molecule binding to p38alpha and derive a detailed thermodynamic reaction pathway for SB-203580. The Biacore method reported here provides an efficient way to directly and reproducibly examine dissociation constants, kinetics, and thermodynamics for small molecules binding to p38alpha and possibly other protein kinases. Immobilization in the presence of a stabilizing ligand may further represent a broadly applicable paradigm for creation of highly active biosensor surfaces.
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PMID:A Biacore biosensor method for detailed kinetic binding analysis of small molecule inhibitors of p38alpha mitogen-activated protein kinase. 1471 93

Deoxycytidine kinase (dCK) is a key enzyme in the deoxynucleoside salvage pathway and in the activation of numerous nucleoside analogues used in cancer and antiviral chemotherapy. Recent studies indicate that dCK activity might be regulated through reversible phosphorylation. Here, we report the effects of a large panel of protein kinase inhibitors on dCK activity in the B-leukemia cell line EHEB, both in basal conditions and in the presence of the nucleoside analogue 2-chloro-2'-deoxyadenosine (CdA) which induces activation of dCK. Except staurosporine and H-7 that significantly reduced the activation of dCK by CdA, no specific protein kinase inhibitor diminished basal dCK activity or its activation by CdA. In contrast, genistein, a general protein tyrosine kinase inhibitor, and AG-490, an inhibitor of JAK2 and JAK3, increased basal dCK activity more than two-fold. Two specific inhibitors of the MAPK/ERK pathway, PD-98059 and U-0126, also enhanced dCK activity. These data suggest that the JAK/MAPK pathway could be involved in the regulation of dCK. Moreover, we show that the activity of dCK, raised by CdA, can return to its initial level by treatment with protein phosphatase-2A (PP2A). Accordingly, dCK activity in intact cells increased upon incubation with okadaic acid (OA) at concentrations that should inhibit PP2A, but not protein phosphatase-1. Activation of dCK by protein kinase inhibitors and OA was also observed in CCRF-CEM cells and in chronic lymphocytic leukemia B-lymphocytes, suggesting a general mechanism of post-translational regulation of dCK, which could be exploited to enhance the activation of antileukemic nucleoside analogues.
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PMID:Activation of deoxycytidine kinase by protein kinase inhibitors and okadaic acid in leukemic cells. 1518 21

Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a(-/-) females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a(-/-) oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a(-/-) oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a(-/-) oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a(-/-) mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.
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PMID:Cyclic nucleotide phosphodiesterase 3A-deficient mice as a model of female infertility. 1525 86

Proliferation of vascular smooth muscle cells (VSMCs) is induced by various mitogens through activation of extracellular signal-regulated protein kinase (ERK) pathway. We recently reported that peroxisome proliferator-activated receptor (PPAR)gamma activators such as 15-deoxy-Delta12,14-prostaglandin J2 (15-d-PGJ2) and thiazolidinediones (TZDs) activated MEK/ERK pathway through phosphatidylinositol 3-kinase (PI3-K) and induced proliferation of VSMCs. However, the precise mechanisms of PPARgamma activators-induced activation of PI3-K/ERK pathway have not been determined. We examined whether transactivation of growth factor receptor is involved in this process. Stimulation of VSMCs with 15-d-PGJ2 or TZDs for 15 min induced phosphorylation of ERK1/2 and Akt. 15-d-PGJ2- or TZDs-induced phosphorylation of ERK1/2 and Akt was inhibited by AG1478, an inhibitor of epidermal growth factor receptor (EGF-R) as well as AG1295, an inhibitor of platelet derived growth factor receptor (PDGF-R). 15-d-PGJ2-induced phosphorylation of both EGF-R and PDGF-R. GM6001, a matrix metalloproteinase inhibitor, and PP2, a Src family protein kinase inhibitor, suppressed 15-d-PGJ2- and TZDs-induced phosphorylation of EGF-R and PDGFbeta-R as well as activation of ERK1/2 and Akt. PDGFbeta-R was co-immunoprecipitated with EGF-R, regardless of the presence or absence of 15-d-PGJ2. These data suggest that 15-d-PGJ2 and TZDs activate PI3-K/ERK pathway through Src family kinase- and matrix metalloproteinase-dependent transactivation of EGF-R and PDGF-R. Both receptors seemed to associate constitutively. This novel signaling mechanisms may contribute to diverse biological functions of PPARgamma activators.
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PMID:15-Deoxy-Delta12,14-prostaglandin J2 and thiazolidinediones transactivate epidermal growth factor and platelet-derived growth factor receptors in vascular smooth muscle cells. 1536 66

In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg(2+) over Mn(2+) ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5'-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.
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PMID:Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress. 1546 34

1 Sphingosine-1-phosphate (S1P) is considered a potent mitogen for mesangial cells and activates the classical mitogen-activated protein kinase (MAPK) cascade via S1P receptors. In this study, we show that S1P signalling is rapidly desensitized upon S1P receptor activation. A complete loss of S1P sensitivity occurs after 10 min of S1P pretreatment and remains for at least 8 h. A similar desensitization is also seen with the S1P mimetic FTY720-phosphate, but not with the nonphosphorylated FTY720, nor with sphingosine or ceramide. 2 Prestimulating the cells with extracellular ATP or UTP, which bind to and activate P2Y receptors on mesangial cells, a similar rapid desensitization of the S1P receptor occurs, suggesting a heterologous desensitization of S1P receptors by P2Y receptor activation. Furthermore, adenosine binding to P1 receptors triggers a similar desensitization. In contrast, two other growth factors, PDGF-BB and TGFbeta2, have no significant effect on S1P-induced MAPK activation. 3 S1P also triggers increased inositol trisphosphate (IP3) formation, which is completely abolished by S1P pretreatment but only partially by ATP pretreatment, suggesting that IP3 formation and MAPK activation stimulated by S1P involve different receptor subtypes. 4 Increasing intracellular cAMP levels by forskolin pretreatment has a similar effect on desensitization as adenosine. Moreover, a selective A3 adenosine receptor agonist, which couples to phospholipase C and increases IP3 formation, exerted a similar effect. 5 Pretreatment of cells with various protein kinase C (PKC) inhibitors prior to ATP prestimulation and subsequent S1P stimulation leads to a differential reversal of the ATP effect. Whereas the broad-spectrum protein kinase inhibitor staurosporine potently reverses the effect, the PKC-alpha inhibitor CGP41251, the PKC-delta inhibitor rottlerin and calphostin C show only a partial reversal at maximal concentrations. 6 Suramin, which is reported as a selective S1P3 receptor antagonist compared to the other S1P receptor subtypes, has no effect on the S1P-induced MAPK activation, thus excluding the involvement of S1P3 in this response. 7 In summary, these data document a rapid homologous and also heterologous desensitization of S1P signalling in mesangial cells, which is mechanistically triggered by PKC activation and eventually another staurosporine-sensitive protein kinase, as well as by increased cAMP formation.
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PMID:Heterologous desensitization of the sphingosine-1-phosphate receptors by purinoceptor activation in renal mesangial cells. 1546 46

Adenosine modulates a variety of cellular functions including calcium-dependent exocytosis. Activation of adenosine A(2A) receptor (A(2A)-R) facilitates neurotransmitter release in some cell types, although the underlying mechanisms are not fully understood. In this study, we found that treatment of PC12 cells with the A(2A)-R agonist CGS21680 promotes calcium-evoked secretion of the fusion protein between neuropeptide Y and modified yellow fluorescence protein (NPY-Venus). CGS21680 treatment of PC12 cells transiently increased the phosphorylation of p38 and JNK MAP kinases and Akt, as well as that of ATF2 and CREB, reaching maximal levels at around 10-15 min of CGS21680 treatment. Importantly, pretreatment of PC12 cells with the PI3K inhibitor LY294002, together with the protein kinase A (PKA) inhibitor KT5720, significantly inhibited CGS21680 enhancement of calcium-dependent NPY-Venus release. Moreover, expression of a dominant-negative form of Akt and the PKA inhibitory polypeptide protein kinase inhibitor (PKI) co-operatively inhibited the facilitating effect of CGS21680 on secretion of NPY-Venus. These data suggest that the PI3K-Akt and PKA pathways play a critical role in A(2A)-R-mediated facilitation of calcium-dependent secretion. We also found that CGS21680 treatment promoted recruitment of the NPY-Venus-containing vesicles to the proximity of the plasma membrane at around 10-15 min of CGS21680 treatment, which may in part account for the facilitated secretion by A(2A)-R activation.
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PMID:Adenosine A2A receptor facilitates calcium-dependent protein secretion through the activation of protein kinase A and phosphatidylinositol-3 kinase in PC12 cells. 1566 5

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases, although the mechanism leading to vascular dysfunction is not clear. The aim of this study was to examine the effect of homocysteine (Hcy) on oxi-dative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were challenged for 24 h with Hcy (10 microM-3 mM) in the presence of various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (100 microM), the p38 mito-gen-activated protein kinase inhibitor SB203580 (2.5 microM), the extracellular signal-regulated kinase inhibitor U0126 (2.5 microM), the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) inhibitor JNK inhibitor II (10 microM), and antioxidants alpha-tocopherol (5 microg/mL) and N-acetyl cysteine (NAC, 2 mM). Reactive oxygen species (ROS) were detected using 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride staining, annexin-V phosphatidyl- serine/propidium iodide, and caspase-3 assay. NADPH oxidase and SAPK/JNK signal were evaluated with immunoblotting. Hcy significantly enhanced ROS generation and apoptosis after 24-h incubation. Apocynin prevented Hcy-induced ROS generation but only partially restored Hcy-induced apoptosis. JNK inhibitor II, alpha-tocopherol, and NAC partially reduced Hcy-induced apoptosis, although SB203580 and U0126 had no effect. Immunoblotting analysis confirmed upregulation of NADPH oxidase and SAPK/JNK signaling. Collectively, our results suggested that Hcy may induce oxidative stress and apopto-sis through an NADPH oxidase and/or JNK-dependent mechanism(s).
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PMID:Possible involvement of NADPH oxidase and JNK in homocysteine-induced oxidative stress and apoptosis in human umbilical vein endothelial cells. 1573 81

Previous research from our laboratory has shown a switch-like response to PCB 126 mediated CYP1A1 induction in primary rat hepatocytes and in H4IIE rat hepatoma cells. On a single cell level, cells appear to be either "on" or "off" for CYP1A1 induction at a given dose; some cells never respond to PCB 126. These cells represent a non-responding population. Cells that are switched "on" by PCB 126 display varying levels of induction, much like the dimmer on a light switch. The goal of the present research is to begin to uncover the mechanism for this switch-like response to CYP1A1 induction in H4IIE rat hepatoma cells. The AhR pathway is modulated by multiple co-activators and by phosphorylation. This research focuses on the phosphorylation cascades initiated by PCB 126 and the role they play in CYP1A1 induction. Our research reveals a likely role for protein kinase C (PKC) in this switch response. Inhibition of PKC by H-7 dramatically reduced the percent of cells that express CYP1A1 in response to PCB 126 treatment, as determined by flow cytometry. The effect of H-7 was concentration dependent, decreasing the number of cells expressing CYP1A1 rather than decreasing the level of CYP1A1 in all cells. This finding provides further evidence for the switch-like behavior of CYP1A1 induction and implicates PKC in this response to PCB126. The protein kinase inhibitor, HA-1004, had only a minor effect on CYP1A1 induction. A high-throughput immunoblot screen for 40 proteins revealed the regulation of several proteins/phosphoproteins by PCB 126. Most importantly, two proteins containing phosphoserine/phoshothreonine residues were increased by PCB126 treatment. However, PKC translocation studies and activity studies failed to verify that PCB126 activates PKC. It is possible that constitutive PKC activity is sufficient to maintain phosphorylation of critical components of the AhR pathway. Immunoblotting studies showed that MAP kinases ERK and JNK are not activated by PCB 126 in H4IIE cells and the ERK inhibitor U0126 did not impair CYP1A1 induction. Additional studies are planned to further investigate the role of PKC in the switch-like response to PCB 126.
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PMID:Probing the control elements of the CYP1A1 switching module in H4IIE hepatoma cells. 1608 25

Macrophages express several P2X and P2Y nucleotide receptors and display the phenomenon of ATP-induced P2X7-dependent membrane permeabilization, which occurs through a poorly understood mechanism. Several P2 receptors are known to be coupled to the activation of mitogen-activated protein kinases (MAPKs) and Ca2+ signaling. Here, we use macrophages to investigate the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by nucleotides and the involvement of MAPKs and intracellular Ca2+ concentration in ATP-induced membrane permeabilization. Short-term (5 min) pre-exposure to oxidized ATP (oATP), a P2X7 antagonist that does not inhibit P2X7-associated inward currents or membrane permeabilization, inhibits the activation of ERK1/2 by ATP, ADP, the P2X7 agonist 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP), but not by UTP and UDP. We conclude that macrophages display several P2Y receptors coupled to the ERK1/2 pathway and that oATP antagonizes the action of purine nucleotides, possibly binding to P2X7 and/or other purine-binding P2Y receptors. We also show that BzATP and ATP activate ERK1/2 by two different pathways since ERK1/2 activation by BzATP, but not by ATP, is blocked by the tryrosine kinase inhibitor, genistein, and the Src protein kinase inhibitor, tyrphostin. However, the activation of ERK1/2 by ATP is blocked by the protein kinase C (PKC) inhibitor, chelerythrine chloride. Under the same conditions, membrane permeabilization is not blocked by genistein, tyrphostin, or chelerythrine chloride, indicating that tyrosine kinase, Src protein kinase, and PKC are not required for pore opening. Membrane permeabilization is independent of ERK1/2 activation since chelerythrine, or short-term exposure to oATP or PD98059, efficiently block ERK1/2 activation without inhibiting membrane permeabilization. In addition, membrane permeabilization is not inhibited by SB203580 and SB202190, two inhibitors of p38 MAPK, nor by intracellular BAPTA, which blocks ATP-induced Ca2+ signals. These results suggest that multiple P2 receptors lead to ERK1/2 activation, that ligation of the same receptors by agonists with different affinities can lead to differential stimulation of separate pathways, and that MAPKs and intracellular Ca2+ fluxes are independent of P2X7-associated pore formation.
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PMID:Activation of ERK1/2 by extracellular nucleotides in macrophages is mediated by multiple P2 receptors independently of P2X7-associated pore or channel formation. 1634 Dec 34


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