Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction events. The A-kinase anchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes with N-methyl-d-aspartic acid or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that residues 315-360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315-360 region of AKAP79 can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a peptide encompassing residues 330-357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities with other proteins that serve to coordinate PP2B localization and activity.
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PMID:Mapping the protein phosphatase-2B anchoring site on AKAP79. Binding and inhibition of phosphatase activity are mediated by residues 315-360. 1235 62

The role of glutamate receptors (GluR) in the signal pathways of the retina is widely recognized. Photoreceptors make synaptic contact with functionally different classes of bipolar cells. The OFF-type bipolar cells mediate light offset-evoked responses and use ionotropic alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)- or kainate-type GluRs, whereas bipolars involved in the ON-pathway use the metabotropic GluR6. This dichotomy predicts a defined expression pattern of AMPA-type GluRs and mGluR6 in bipolar cell classes. This hypothesis was tested by performing immunocytochemical double labeling studies combining GluR-specific antibodies with markers specific for the diverse bipolar cell populations in the rat retina. AMPA-type receptors are composed of combinations of four types of subunits, GluR1-4. GluR1 is expressed by a few somata in the outer part of the inner nuclear layer (INL). Sparse colocalization with any of the bipolar markers used could be established. In contrast, GluR2 is expressed by many of the somata in the outer zone of the INL. At the transcript level, in situ hybridizations demonstrated abundant GluR2 expression over the complete width of the INL. In contrast to our expectations, approximately 70% of the somata labeled by the rod ON-bipolar markers protein kinase C (PKC) or Goalpha, colocalized with GluR2. Approximately 90% of the OFF-type bipolar cells, identified as recoverin-positive, showed GluR2 immunoreactivity. At least 40% of the somata that were mGluR6-immunoreactive, a both rod and cone ON-type bipolar marker, were GluR2-immunopositive. Ultrastructurally, examples were observed of GluR2 localization in bipolar processes with labeling outside the actual compartment associated with the synaptic complex of the rod terminal. No specific antibody was available against GluR3, but 74% of the PKC-positive cells were GluR2/3-positive. GluR4 did not show a somatic localization making double labeling impossible. On the basis of these results, we conclude that ionotropic GluRs are expressed by rod ON-type bipolar cells (PKC- or Goalpha-immunoreactive), and by cone ON- and OFF-type bipolars based on a colocalization with nearly all of the present recoverin-positive somata. Our observations show that the functional dichotomy in ON- and OFF-type bipolars is not reflected in a matching expression pattern of ionotropic and metabotropic GluRs. This finding raises the intriguing possibility that the AMPA-type GluRs are, in an as yet unclear manner, involved in the ON signaling pathways of rods and cones.
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PMID:Expression of AMPA-type glutamate receptor subunit (GluR2) in ON-bipolar neurons in the rat retina. 1245 83

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKC gamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKC gamma in cultured chick retinal neurons. Pull-down assays showed that native PKC gamma binds the GluR4 C-terminal membrane-proximal region, and recombinant PKC gamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKC gamma. The GluR4 C-terminal segment that interacts with PKC gamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKC gamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKC gamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKC gamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.
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PMID:Protein kinase C gamma associates directly with the GluR4 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit. Effect on receptor phosphorylation. 1247 Oct 40

The excitotoxic cascade may represent an important pathway leading to brain damage and cerebral palsy. Brain lesions induced in newborn mice by ibotenate (acting on N-methyl-D-aspartate receptors) and by S-bromowillardiine (acting on alpha-3-amino-hydroxy-5-methyl-4-isoxazole propionic acid and kainate receptors) mimic some aspects of white matter cysts and transcortical necrosis observed in human perinatal brain damage. Fructose 1,6-biphosphate (FBP) is a high-energy glycolytic pathway intermediate which, in therapeutic doses, is non-toxic and neuroprotective in hypoxic-ischemic models of brain injury. Mechanisms of action include modulation of intracellular calcium through phospholipase C (PLC) activation. The goal of this study was to determine the neuroprotective effects of FBP in a mouse model of neonatal excitotoxic brain injury. Mice that received intraperitoneal FBP had a significant reduction in size of ibotenate-induced (80% reduction) or S-bromowillardiine-induced (40% reduction) cortical plate lesions when compared with control animals. Studies of fragmented DNA and cleaved caspase 3 confirmed the survival promoting effects of FBP. FBP had no detectable effect on excitotoxic white matter lesions. The effects of FBP were antagonized by co-administration of PLC, protein kinase C or mitogen-associated protein kinase inhibitors but not by protein kinase A inhibitor. A moderate, transient cooling of pups immediately after the insult extended the therapeutic window for FBP, as FBP administered 24 h after ibotenate was still significantly neuroprotective in these pups. This data extends the neuroprotective profile of FBP in neonatal brain injury and identifies gray matter lesions involving N-methyl-D-aspartate receptors as a major target for this promising drug.
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PMID:Fructose-1,6-biphosphate prevents excitotoxic neuronal cell death in the neonatal mouse brain. 1258 34

We used immunoblot analysis to investigate the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-receptor glutamate receptor-1 (GluR1) and related protein kinases in rat hippocampus on postnatal days (PND) 1-28. Total GluR1 expression increased up to PND 9, and stayed high thereafter. The proportions of the forms of GluR1 phosphorylated at serines (S) 845 and S831, which were both high at birth, decreased at different rates: phosphorylated S831 decreased rapidly after PND 7, and was almost zero after PND 21, while phosphorylated S845 decreased slowly, and after PND 14 stayed at one third of its PND 1 level. The expression patterns of cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), but not that of calcium/calmodulin-dependent protein kinase II, were similar to those of the forms of GluR1 phosphorylated at S845 and S831, respectively. Thus, the status of GluR1 phosphorylation, through PKA and PKC modulation, may contribute to the development of hippocampal synaptic plasticity.
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PMID:Differential phosphorylation at serine sites in glutamate receptor-1 within neonatal rat hippocampus. 1267 39

Alterations in motor response that complicate levodopa treatment of Parkinson's disease appear to involve sensitization of striatal ionotropic glutamate receptors. Since protein kinase C (PKC)-mediated phosphorylation regulates glutamatergic receptors of the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) subtype and has been linked to several forms of behavioral plasticity, activation of PKC signaling in striatal spiny neurons may also contribute to the motor plasticity changes associated with chronic levodopa therapy. To evaluate this possibility, we sought to augment PKC signaling by using Herpes Simplex Virus type 1 vectors (pHSVpkcDelta) to directly transfer the catalytic domain of the PKCbetaII gene into striatal neurons of parkinsonian rats. Microinjection of pHSVpkcDelta vectors lead to the persistent expression of PkcDelta (35% loss over 21 days) in medium spiny neurons together with an increase in serine 831 phosphorylation on AMPA receptor GluR1 subunits and hastened the appearance of the shortened response duration produced by chronic levodopa treatment (P<0.05). In pHSVpkcDelta-infected animals, intrastriatal injection of the PKC inhibitor NPC-15437 (1.0 microg) attenuated both the increased GluR1 phosphorylation (P<0.01) and the accelerated onset of the levodopa-induced response modifications (P<0.01). However, in rats that received levodopa treatment for 21 days without the gene transfer, intrastriatal NPC-15437 had no effect on the response shortening or on GluR1 S831 phosphorylation. The results suggest that an increase in PKC-mediated signaling, including, in part, phosphorylation of AMPA receptors, on striatal spiny neurons may be sufficient to promote the initial appearance, but not necessary the ultimate expression, of the levodopa-induced motor response changes occurring in a rodent model of the human motor complication syndrome.
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PMID:Gene transfer of constitutively active protein kinase C into striatal neurons accelerates onset of levodopa-induced motor response alterations in parkinsonian rats. 1269 33

The present study aimed at understanding the effect of FR236924, a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on hippocampal synaptic transmission in both the in vitro and in vivo systems. FR236924 increased the rate of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, triggered by nicotine in CA1 pyramidal neurons of rat hippocampal slices, that is inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor or alpha-bungarotoxin, an inhibitor of alpha7 acetylcholine (ACh) receptors. FR236924 stimulated glutamate release from rat hippocampal slices and in the hippocampus of freely behaving rats, and the effect was also inhibited by GF109203X or alpha-bungarotoxin. FR236924 induced a transient huge potentiation followed by a long-lasting potentiation in the slope of field excitatory postsynaptic potentials recorded from the CA1 region of rat hippocampal slices, and the latter effect was blocked by GF109203X or alpha-bungarotoxin. Likewise, the compound persistently facilitated hippocampal synaptic transmission in the CA1 region of the intact rat hippocampus. It is concluded from these results that FR236924 stimulates glutamate release by functionally targeting presynaptic alpha7 ACh receptors on the glutamatergic terminals under the influence of PKC, responsible for the facilitatory action on hippocampal synaptic transmission. This may provide evidence for a link between cis-unsaturated free fatty acids and presynaptic alpha7 ACh receptors in hippocampal synaptic plasticity.
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PMID:The linoleic acid derivative FR236924 facilitates hippocampal synaptic transmission by enhancing activity of presynaptic alpha7 acetylcholine receptors on the glutamatergic terminals. 1556 36

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nM, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.
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PMID:Blocking tumor cell migration and invasion with biphenyl isoxazole derivative KRIBB3, a synthetic molecule that inhibits Hsp27 phosphorylation. 1623 46

Kainate receptors (KARs) are widely expressed the basal ganglia. In this study, we used electron microscopic immunocytochemistry and whole-cell recording techniques to examine the localization and function of KARs in the rat globus pallidus (GP). Dendrites were the most common immunoreactive elements, while terminals forming symmetric or asymmetric synapses and unmyelinated axons comprised most of the presynaptic labeling. To determine whether synaptically released glutamate activates KARs, we recorded excitatory postsynaptic currents (EPSCs) in the GP following single-pulse stimulation of the internal capsule. 4-(8-Methyl-9H-1,3-dioxolo[4,5 h]{2,3}benzodiazepine-5-yl)-benzenamine hydrochloride (GYKI 52466, 100 microm), an alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, reduced but did not completely block evoked EPSCs. The remaining EPSC component was mediated through activation of KARs because it was abolished by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), an AMPA/KAR antagonist. The rise time (10-90%) and decay time constant (tau) for those EPSCs were longer than those of AMPA-mediated EPSCs recorded before GYKI 52466 application. KAR activation inhibited EPSCs. This inhibition was associated with a significant increase in paired-pulse facilitation ratio, suggesting a presynaptic action of KAR. KAR inhibition of EPSCs was blocked by the G-protein inhibitor, N-ethylmaleimide (NEM), or the protein kinase C (PKC) inhibitor calphostin C. Our results demonstrate that KAR activation has dual effects on glutamatergic transmission in the rat GP: (1) it mediates small-amplitude EPSCs; and (2) it reduces glutamatergic synaptic transmission through a presynaptic G-protein coupled, PKC-dependent, metabotropic mechanism. These findings provide evidence for the multifarious functions of KARs in regulating synaptic transmission, and open up the possibility for the development of pharmacotherapies to reduce the hyperactive subthalamofugal projection in Parkinson's disease.
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PMID:Localization and function of pre- and postsynaptic kainate receptors in the rat globus pallidus. 1642 Apr 45

The propagation of Ca2+ waves in a network of microglial cells, after its initiation by glutamate, is mediated by purinergic transmission. In this study, we investigated the mechanisms by which glutamate releases ATP from cultured spinal cord microglia. The 4-fold increase in ATP release from microglia in response to glutamate (0.5 mM) was blocked by alpha-aminohydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptor antagonist 6-cyano-7-nitroguinoxaline-2,3-dione and specific AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) but not by N-methyl-d-aspartic acid or metabotropic glutamate receptor antagonists. Glutamate acting on AMPA receptors evoked an ATP release that was blocked by antagonizing the rise in intracellular Ca2+ as a result of its release from internal stores as well as by antagonizing protein kinase C with chelerythrine. Glutamate-stimulated ATP release was significantly antagonized by the cystic fibrosis transmembrane conductance regulator (CFTR) blockers flufenamic acid and glibenclamide. A role for the CFTR was further confirmed using microglia from CFTR knockout mice, which released significantly less ATP than microglia from control wild-type mice in response to glutamate. Use of 6-methoxy-1-(3-sulfopropyl)quinolinium fluorescence assay revealed functional CFTR in microglia. These observations suggest that glutamate acted on microglial AMPA receptors to stimulate release of Ca2+ from intracellular stores as well as a Ca2+-dependent isoform of protein kinase C, which then acts to trigger release of ATP with the CFTR acting as a regulator of the ATP release process, perhaps through another channel or transporter.
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PMID:Purine release from spinal cord microglia after elevation of calcium by glutamate. 1676 Mar 62


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