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 describe the transient expression of the rat skeletal muscle muI Na+ channel in human embryonic kidney (HEK 293) cells. Functional channels appear at a density of approximately 30 in a 10 microns 2 patch, comparable to those of native excitable cells. Unlike muI currents in oocytes, inactivation gating is predominantly (approximately 97%) fast, although clear evidence is provided for noninactivating gating modes, which have been linked to anomalous behavior in the inherited disorder hyperkalemic periodic paralysis. Sequence-specific antibodies detect a approximately 230 kd glycopeptide. The majority of molecules acquire only neutral oligosaccharides and are retained within the cell. Electrophoretic mobility on SDS gels suggests the molecules may acquire covalently attached lipid. The channel is readily phosphorylated by activation of the protein kinase A and protein kinase C second messenger pathways.
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PMID:muI Na+ channels expressed transiently in human embryonic kidney cells: biochemical and biophysical properties. 131 19

The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.
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PMID:Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta. 173 21

Glutamate-gated ion channels mediate most excitatory synaptic transmission in the mammalian central nervous system and play major roles in synaptic plasticity, neuronal development, and in some neuropathological conditions. Recent studies have suggested that protein phosphorylation of neuronal glutamate receptors by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) may regulate their function and play a role in some forms of synaptic plasticity. To test whether these protein kinase effects are due to direct phosphorylation of the receptors and to further examine the sites and mechanisms by which the receptors are modulated, we transiently expressed recombinant glutamate receptors in HEK-293 cells and studied their biochemical and biophysical properties. Our results indicate that the kainate-preferring receptor GluR6 is phosphorylated by PKA, primarily on a single serine in the proposed major intracellular loop. Moreover, using the whole cell patch clamp recording technique, we have shown that phosphorylation at this site increases the amplitude of the GluR6-mediated glutamate current without significantly altering its dose-response, current-voltage relation or desensitization kinetics. In other experiments, we have demonstrated that the NMDA receptor subunit NR1 is phosphorylated by PKC on several distinct sites, and most of these sites are located within a single alternatively spliced exon in the C-terminal domain. These findings suggest that RNA splicing can regulate NMDA receptor phosphorylation and that, contrary to the previously proposed membrane topology model, the NR1 C-terminus is intracellular. Furthermore, in HEK-293 cells co-transfected with NR2A and NR1 subunits containing the C-terminal exon with the PKC phosphorylation sites, our preliminary studies indicate that the NMDA-evoked current is potentiated by intracellular PKC. We are currently examining PKC effects on the NMDA-evoked current responses of mutant NR1 receptors that lack the C-terminal phosphorylation sites. These studies provide evidence that glutamate receptors are directly phosphorylated and functionally modulated by protein kinases. Moreover, by identifying phosphorylation sites within the receptor proteins, our results provide information about the structure and membrane topology of these receptors.
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PMID:Glutamate receptor modulation by protein phosphorylation. 753 May 47

We have examined the regulation of neuronal nitric oxide synthase (NOS) by phosphorylation with cyclic-GMP (PKG) and cyclic-AMP-dependent (PKA) protein kinases. In vitro phosphorylation studies indicate that both PKG and PKA phosphorylate NOS on a single site. Phosphoamino-acid analysis and peptide mapping demonstrate that phosphorylation by either cyclic-nucleotide kinase occurs on a similar serine residue. Phosphorylation of purified NOS by either PKG or PKA diminishes catalytic activity. Stimulation by 8-Br-cGMP of HEK-293 cells stably transfected with the cDNA for neuronal NOS (293.NOS cells) results in phosphorylation of immunoprecipitated NOS. Incubation of 293-NOS cells with 8-bromo-cGMP or dibutyryl-cAMP reduces nitrite release in response to stimulation with calcium ionophore A23187. Phosphorylation-induced decreases in NOS activity may counterbalance and modulate NOS activating signals.
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PMID:Cyclic nucleotide dependent phosphorylation of neuronal nitric oxide synthase inhibits catalytic activity. 753 10

The actions of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) on cellular signalling were studied in human embryonal kidney 293 (HEK 293) cells stably transfected with the cloned human GLP-1 receptor. The cloned GLP-1 receptor showed a single high-affinity binding site (Kd = 0.76 nM). Binding of GLP-1(7-36)amide stimulated cAMP production in a dose-dependent manner (EC50 = 0.015 nM) and caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The latter effect reflected Ca(2+)-induced Ca2+ release and was suppressed by ryanodine. We propose that the ability of GLP-1(7-36)amide to increase [Ca2+]i results from sensitization of the ryanodine receptors by a protein kinase A dependent mechanism.
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PMID:Stimulation of cloned human glucagon-like peptide 1 receptor expressed in HEK 293 cells induces cAMP-dependent activation of calcium-induced calcium release. 758 61

The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.
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PMID:Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II. 793 83

Studies carried out with mammals and invertebrates suggest that Ca(2+)-sensitive adenylyl cyclases may be important for neuroplasticity. Long-term potentiation in the hippocampus requires increases in intracellular Ca2+ which are accompanied by elevated cyclic AMP (cAMP). Furthermore, activation of cAMP-dependent protein kinase is required for the late stage of long-term potentiation in the CA1 region of the hippocampus, which is also sensitive to inhibitors of transcription. Therefore, some forms of synaptic plasticity may require coordinate regulation of transcription by Ca2+ and cAMP. In this study, we demonstrate that the expression of type I adenylyl cyclase in HEK-293 cells allows Ca2+ to stimulate reporter gene activity mediated through the cAMP response element. Furthermore, simultaneous activation by Ca2+ and isoproterenol caused synergistic stimulation of transcription in HEK-293 cells and cultured neurons. We propose that Ca2+ and neurotransmitter stimulation of type I adenylyl cyclase may play a role in synaptic plasticity by generating optimal cAMP signals for regulation of transcription.
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PMID:Type I adenylyl cyclase functions as a coincidence detector for control of cyclic AMP response element-mediated transcription: synergistic regulation of transcription by Ca2+ and isoproterenol. 796 63

Hormones can regulate cardiac L-type Ca2+ channels via cAMP-dependent protein kinase (PKA) phosphorylation. However, regulation of the cloned L-type Ca2+ channel has been difficult to demonstrate conclusively. We stably transfected a human embryonic kidney (HEK-293) cell with the cardiac alpha 1 and beta 2 subunits, then examined PKA modulation of the Ca2+ current. Although forskolin did not increase basal Ca2+ current, the PKA inhibitors, H-89 and Rp-cAMPS, could inhibit basal current. We reversed H-89 inhibition with either forskolin or okadaic acid. We conclude that the channel was phosphorylated under basal conditions, and that inhibition of PKA allowed dephosphorylation. These studies demonstrate that reversible PKA regulation of cloned Ca2+ channels can be studied in HEK-293 cells.
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PMID:Regulation of the cloned L-type cardiac calcium channel by cyclic-AMP-dependent protein kinase. 814 62

The family of beta-amyloid protein precursors (APP) can be processed via several alternative proteolytic pathways. Some generate potentially amyloidogenic APP derivatives, whereas others preclude the formation of such fragments. The cellular mechanisms regulating the relative activities of these pathways are thus important in determining the factors contributing to the formation of amyloidogenic APP derivatives. In order to investigate whether cell-surface receptor activity can regulate APP processing, HEK 293 cell lines stably expressing human muscarinic acetylcholine receptors (mAChR; subtypes m1, m2, m3, m4) were stimulated with the muscarinic agonist carbachol, and the release of APP derivatives was measured. Carbachol increased the release of large amino-terminal APP-fragments 4- to 6-fold in cell lines expressing the m1 or m3 receptors but not in those expressing m2 or m4 subtypes. This increase was blocked by various protein kinase inhibitors and mimicked by phorbol esters, indicating that it is mediated by protein kinase activation, presumably by protein kinase C (PKC). To determine whether additional cell-surface receptor types linked to this signal transduction pathway could also regulate APP processing, we stimulated differentiated PC-12 cells with bradykinin and found that this neuropeptide also increased the secretion of amino-terminal APP derivatives. We next investigated the possibility that neuronal depolarization might affect APP processing in mammalian brain. Electrically stimulated rat hippocampal slices released two times more amino-terminal APP derivatives than unstimulated control slices. This release increased with increasing stimulation frequencies in the physiological firing range of hippocampal pyramidal cells, and was blocked by tetrodotoxin. These results suggest that, in brain, APP processing is regulated by neuronal activity.
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PMID:Receptor-coupled amyloid precursor protein processing. 823 69

We have previously amplified cDNA subfragments of protein-tyrosine-kinases (PTKs) by using the polymerase chain reaction (PCR) and specific sets of oligonucleotide primers derived from nucleotide sequences of their kinase domain. In this study we have used a more directed approach to identify new members of the EPH/elk-family by PCR of human embryonic cDNA: we utilized oligonucleotide primers specifically designed to a highly conserved N-terminal motif and the kinase region of EPH/elk-PTKs in RNA-PCRs. The 5' and 3' elongation of the primary PCR product was achieved by the RACE (rapid amplification of cDNA ends)-technique. Sequence analysis of 3.8 kb of overlapping PCR products allowed to identify a novel receptor-PTK, HEK 2 (human embryo kinase 2), as an additional member of this family, without the need to screen a cDNA library. This approach should be useful for the rapid isolation of other PTK-genes as well. Analysis of genomic DNA placed HEK 2 on chromosome 3. Northern blot analysis demonstrated the expression of a 4.6 kb HEK 2-mRNA in lung, brain, pancreas, liver, placenta, kidney, skeletal muscle, heart and several human cells. In a protein kinase assay with HEK 2-specific immunoprecipitates from the human epidermoid carcinoma cell line A431, a protein of 130 kDa was found phosphorylated.
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PMID:PCR mediated detection of a new human receptor-tyrosine-kinase, HEK 2. 839 71


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