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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used monolayers of parental 3T3 fibroblasts and 3T3 cells expressing transfected cell adhesion molecules (CAMs, NCAM, N-cadherin, or L1) as a culture substrate for cerebellar neurons. Previous studies suggest that the transfected CAMs promote neurite outgrowth by activating a second messenger pathway within the responding neuron that involves influx of calcium into neurons as a consequence of activation of an FGF receptor. The same neurite outgrowth response can be induced by FGF or a number of agents that directly activate defined steps in the CAM signaling pathway. In the present study we show that the neurite outgrowth stimulated by the above three CAMs, FGF, arachidonic acid (AA), and K+ depolarization can be abolished by the Ca2+/calmodulin-dependent (CaM) kinase inhibitor, KN-62. We also demonstrate that neurite outgrowth over astrocytes, which represent a more physiologically relevant cellular substrate, can be substantially inhibited by a number of agents that block the CAM signaling pathway, including KN-62. However, neurite outgrowth induced by activation of protein kinase A is unaffected by inhibition of CaM kinase activity as is basal neurite outgrowth over 3T3 monolayers or a polylysine/laminin substrate. These results suggest that CaM kinase activity is specifically required downstream of calcium influx in the CAM and FGF signaling pathway leading to axonal growth.
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PMID:A Ca2+/calmodulin kinase inhibitor, KN-62, inhibits neurite outgrowth stimulated by CAMs and FGF. 759 59

gamma-Aminobutyric acid type A receptor subunits (GABAA) can be divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cGMP-dependent protein kinase (PKG) and multifunctional calcium/calmodulin-dependent protein kinase (CAM KII). Both PKG and CAM KII phosphorylated a purified beta 1 subunit fusion. Both of these kinases phosphorylated serine 409 within the beta 1 subunit; in addition, CAM KII also phosphorylated serine 384 as determined by site-specific mutagenesis. Fusion proteins of the major intracellular domains of the gamma 2S and gamma 2L subunits were produced. These proteins differ by 8 amino acids (LLRMFSFK). Both the gamma 2L and gamma 2S fusion proteins were excellent substrates of CAM KII. However, the gamma 2L fusion protein was phosphorylated to higher stoichiometry due to the phosphorylation of serine 343 within this 8-amino acid insertion. Both the gamma 2L and gamma 2S subunits were phosphorylated on common residues by CAM KII identified as serine 348 and threonine 350. These results identify specific sites of phosphorylation for CAM KII and PKG within GABAA receptor subunits, suggesting a role for these two kinases in modulating GABAA receptor function in vivo.
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PMID:Differential phosphorylation of intracellular domains of gamma-aminobutyric acid type A receptor subunits by calcium/calmodulin type 2-dependent protein kinase and cGMP-dependent protein kinase. 802 73

The gamma 2 subunit of the GABA receptor (GABAA-R) is alternatively spliced. The long variant (gamma 2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336-351 of the intracellular loop of the gamma 2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The Km values for PKC- and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 microM, respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the gamma 2L subunit has been shown to be necessary for ethanol potentiation of the GABAA-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.
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PMID:Ca2+/calmodulin-dependent protein kinase II and protein kinase C phosphorylate a synthetic peptide corresponding to a sequence that is specific for the gamma 2L subunit of the GABAA receptor. 839 May 66

The possible role of Ca++/calmodulin-dependent protein kinase II (CAM-K-II) in the nicotinic activation of tyrosine hydroxylase in intact cultured bovine adrenal chromaffin cells has been investigated. Over the concentration range 3-30 microM, KN62, a specific CAM-K-II inhibitor, inhibited basal tyrosine hydroxylase activity and the activity stimulated by nicotine or K+ depolarisation. KN04, a structural analogue of KN62 which does not inhibit CAM-K-II, produced an identical concentration-dependent inhibition of basal and nicotine-stimulated tyrosine hydroxylase activity. Another CAM-K-II inhibitor, KN93, also inhibited nicotine and K+ stimulation of tyrosine hydroxylase activity; however, an inactive analogue of KN93, KN92, mimicked these effect. The results suggest that the inhibition of nicotine- and K+-stimulated tyrosine hydroxylase activity by KN62 and KN93 is not due to their ability to inhibit CAM-K-II.
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PMID:The Ca++/calmodulin-dependent protein kinase II inhibitors KN62 and KN93, and their inactive analogues KN04 and KN92, inhibit nicotinic activation of tyrosine hydroxylase in bovine chromaffin cells. 866 Mar 26

Membrane depolarization, or agents which increase intracellular calcium, elicit transcriptional activation of tyrosine hydroxylase (TH). In this study we analyze the factors involved in the regulation of the TH promoter by a calcium ionophore. PC12 cells were transiently transfected with plasmids containing wild type or mutated 5' flanking sequences of the rat TH gene, fused to bacterial chloramphenicol acetyl transferase (CAT). Point mutations introduced into the consensus cAMP-regulatory element (CRE) abolished the induction of CAT by ionomycin indicating that it is essential for mediating the calcium response. An intact and functional AP1 site did not confer calcium inducibility when the CRE/CaRE sequence was mutated. The extent and kinetics of the increase in intracellular calcium as well as the induction of CAT activity under the control of TH promoter by ionomycin were similar in PC12 cells and in the A123.7, protein kinase A (PKA) deficient cell line. In both cell lines addition of ionomycin rapidly increased the phosphorylation of transcription factor CREB at Ser-133. These results suggest that the activation of TH transcription by ionomycin does not require PKA. However, KN62 an inhibitor of Ca2+/calmodulin dependent (CaM) kinases prevented the induction indicating possible involvement of CAM kinases in the calcium response.
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PMID:Promoter elements and second messenger pathways involved in transcriptional activation of tyrosine hydroxylase by ionomycin. 871 52

Chronic feeding of ethanol to rats results in disorganization of the keratin intermediate filament network within hepatocytes. Previous studies from this laboratory have shown that intermediate filament organization in cultured cells is related to the phosphorylation state of the proteins. Therefore, we have examined the phosphorylation state of hepatocyte keratins from control and ethanol-fed rats. Feeding ethanol to rats results in dephosphorylation of one site on keratin 8 and one site on keratin 18 at all time points beginning with 6 weeks of ethanol treatment. Dephosphorylation was detected by phosphate analysis and by two-dimensional electrophoresis in which a change in isoelectric point of keratins from ethanol-fed rats was observed. These observations indicate that dephosphorylation of keratins in ethanol-fed animals may be an early step in alcoholic hepatitis which has occurred by 6 weeks of ethanol treatment. To further characterize keratin dephosphorylation in ethanol-fed rats, we used 31P NMR spectroscopy to classify the dephosphorylation site(s). Hepatocyte keratins were purified and solubilized in 9.5 M urea, 10 mM Tris-Cl, pH 8.1. 31P NMR spectra were obtained at 109 MHz, in 10 mm tubes at 30 degrees C. Samples of hepatocyte keratins were phosphorylated with A-kinase, protein kinase C, casein kinase II or Ca/CAM kinase and these samples were analyzed by 31P NMR spectroscopy. The resulting spectra were used as standards to compare the 31P chemical shifts of the resonances produced by these kinases with the phosphorus resonances of control and experimental samples. The 31P NMR spectrum of control hepatocyte keratins shows three resonances at 0.7, 4 and 5 ppm. In vitro phosphorylation by A-kinase produces a resonance at 4 ppm which is distinctly different from the resonance produced by each of the other kinases. In hepatocyte keratins from ethanol-fed animals, the resonance at 4 ppm was missing from the spectrum. These observations indicate that the keratin site that is dephosphorylated in ethanol-fed rats is characterized by the same 31P chemical shift as the keratin site that is phosphorylated by A-kinase.
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PMID:Site-specificity of ethanol-induced dephosphorylation of rat hepatocyte keratins 8 and 18: A 31P NMR study. 882 32

Ca ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic and endoplasmic reticulum (SER). Phospholamban was first identified as a phosphoprotein in cardiac myocytes. Functional properties of phospholamban by steady-state and presteady-state kinetic studies of Ca pump ATPase suggest that phospholamban functions as an inhibitory co-factor for cardiac Ca ATPase (SERCA 2). Protein kinase A-catalyzed phosphorylation of phospholamban results in the dissociation of phospholamban from the Ca ATPase, thus augmenting the ATPase activity. Phospholamban is found as a homo-pentamer, formed from subunits of 6080 Da in size. PKA-catalyzed and CAM kinase- catalyzed phosphorylation residues (Ser 16 and Thr 17) are located in the N-terminal cytoplasmic domain, whereas the C-terminal 22 residues are extremely hydrophobic and are considered to be embedded in the SR membrane. At least three kinds of Ca ATPase have been found. SERCA 1 is expressed in fast-twitch skeletal muscle, while the SERCA 2 gene encodes two alternatively spliced products, SERCA 2a and 2b. SERCA 2a is expressed in cardiac and slow-twitch skeletal muscles; SERCA 2b in smooth muscle and non-muscle tissues. SERCA 3 is expressed in a broad variety of muscle and non-muscle tissues. In vitro expression systems revealed that the functional properties of Ca transport of SERCA 2 are identical to SERCA 1, but not SERCA 3. In particular, the Ca affinity for Ca transport of SERCA 1 or 2 is lowered by co-expression with phospholamban, whereas that of SERCA 3 is not. Identification of the interaction sites of phospholamban and SERCA 2 helps defining the molecular mode of interaction between the two proteins. Photoactivated cross-linking studies indicated that potential binding residues are located just downstream of the active ATPase site (Asp 351) of SERCA 2, but SERCA 3 is devoid of this sequence. If a chimeric Ca ATPase (CH2) is made from SERCA 2 and 3, in which the SERCA 3 region corresponding to the phospholamban-binding sequence of SERCA 2 is introduced into the remainder of the SERCA 2 molecule, then the interaction with phospholamban is lost. These results suggest that this region of SERCA 2 contains amino acids which are involved in the interaction with phospholamban. By site-directed mutagenesis of amino acids of this region, we were able to show that 6 residues, Lys-Asp-Asp-Lys-Pro-Val402, of SERCA 2 are functionally important for the interaction. When the chimera CH2 was mutated back to SERCA 2 type, mutated CH2 containing these 6 residues of SERCA 2 restored the interaction with phospholamban. Altogether, these 6 residues of SERCA 2 represent the interaction sites for phospholamban. Mutagenesis studies of phospholamban also demonstrated that the hydrophilic, cytoplasmic region of phospholamban contains a potential binding site for SERCA 2. We therefore conclude that the functional interaction between the two proteins occurs in the cytoplasmic region.
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PMID:SR Ca(2+)-ATPase/phospholamban in cardiomyocyte function. 895 64

Phosphoenolpyruvate carboxylase (PEPC) genes and cDNA sequences have so far been isolated from a broad range of angiosperm but not from gymnosperm species. We constructed a cDNA library from seedlings of Norway spruce (Picea abies) and identified cDNAs coding for PEPC. A full-length PEPC cDNA was sequenced. It consists of 3522 nucleotides and has an open reading frame (ORF) that encodes a polypeptide (963 amino acids) with a molecular mass of 109551. The deduced amino acid sequence revealed a higher similarity to the C3-form PEPC of angiosperm species (86-88%) than to the CAM and C4 forms (76-84%). The putative motif (Lys/Arg-X-X-Ser) for serine kinase, which is conserved in all angiosperm PEPCs analysed so far, is also present in this gymnosperm sequence. Southern blot analysis of spruce genomic DNA under low-stringency conditions using the PEPC cDNA as a hybridization probe showed a complex hybridization pattern, indicating the presence of additional PEPC-related sequences in the genome of the spruce. In contrast, the probe hybridized to only a few bands under high-stringency conditions. Whereas this PEPC gene is highly expressed in roots of seedlings, a low-level expression can be detected in cotyledons and adult needles. A molecular phyiogeny of plant PEPC including the spruce PEPC sequence revealed that the spruce PEPC sequence is clustered with monocot and dicot C3- form PEPCs including the only dicot C4 form characterized so far.
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PMID:Molecular characterization of a phosphoenolpyruvate carboxylase in the gymnosperm Picea abies (Norway spruce). 898 May 43

Adhesion molecules mediate inflammatory myocardial injury after ischemia/reperfusion. Cytokine release and hypoxia are features of acute ischemia that may influence expression of these molecules. Accordingly, we studied intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) responses to cytokines and acute hypoxia in cultured myocardial cells. Northern blot analysis and immunoassay showed that the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha stimulated concentration-dependent increases in ICAM and VCAM mRNA and protein. In both cardiac myocytes and fibroblasts, pretreatment with a specific inhibitor of nuclear transcription factor-kappaB (NF-kappaB) prevented cytokine induction of both molecules. We also found that inhibition of tyrosine kinase and p38/RK (stress-activated protein kinase) pathways prevented IL-1beta-induced ICAM and VCAM protein synthesis, whereas extracellular signal-regulated protein kinase (ERK1/ERK2) inhibition did not. Neither hypoxia (0% O2 for 6 hours) alone nor hypoxia/reoxygenation had any significant effect on ICAM and VCAM mRNA. However, hypoxia did enhance IL-1beta-induced ICAM mRNA expression in myocytes. As a possible mechanism of this synergistic action on CAM expression, hypoxia induced a time-dependent increase in the DNA binding activity of both NF-kappaB and activator protein-1 (AP-1), two transcription factors important for cell adhesion molecule expression. In contrast to the enhanced ICAM mRNA induced by IL-1beta during hypoxia, however, protein levels for this adhesion molecule were unchanged beyond IL-1beta-stimulated levels, suggesting posttranscriptional and/or posttranslational control mechanisms. We conclude that cytokines regulate ICAM and VCAM mRNA and protein in both cardiac myocytes and fibroblasts. Furthermore, adhesion molecule induction requires translocation of at least two transcription factors, NF-kappaB and AP-1.
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PMID:Expression and regulation of adhesion molecules in cardiac cells by cytokines: response to acute hypoxia. 952 62

The length of eukaryotic cilia and flagella depends on the cell cycle-regulated assembly and disassembly of at least 9 doublet and 2 central microtubules, their associated proteins, and the surrounding membrane. In light-synchronized Chlamydomonas cells, flagella assembled to 10-14 microm in length near the beginning of the light period and they disassembled prior to cell division, during the dark period. Flagella on light-synchronized pf18 Chlamydomonas mutants grew to 10-12 microm near the beginning of the light period but shortened by 50% or more by the end of the light period. Flagellar length was cell-cycle regulated: when flagella were amputated at various times during the light period, new flagella regenerated to the lengths of control cells at that time of the light cycle. The later in the cycle pf18 cells were deflagellated, the shorter were the regenerated flagella. Flagellar shortening was not affected, in either pf18 or wild-type (wt) cells, by inhibitors of protein synthesis or of microtubule assembly, so flagellar length cannot depend on protein turnover. Shortening in pf18 was attenuated by Li+, which stimulated flagellar growth in wt cells, by red light, by protein kinase inhibitors, and by the Ca2+ channel blockers La3+ and Cd2+. Shortening was increased by cAMP, Na+, K+, and EGTA. Ca2+-CAM blockers did not affect pf18 shortening but they increased shortening in wt and fa1 cells. We propose that flagellar length is regulated by a signal transduction pathway that is sensitive to Ca2+ levels and red light.
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PMID:Regulation of flagellar length in Chlamydomonas. 963 11


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