Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phosphorylation of synaptophysin, a major integral membrane protein of small synaptic vesicles, was found to be regulated in a Ca(2+)-dependent manner in rat cerebrocortical slices, synaptosome preparations, and highly purified synaptic vesicles isolated from rat forebrain. K(+)-induced depolarization of slices and synaptosomes prelabeled with 32P-orthophosphate produced a rapid, transient increase in serine phosphorylation of synaptophysin. In synaptosomes, the depolarization-dependent increase in synaptophysin phosphorylation required the presence of external Ca2+ in the incubation medium. The addition of Ca2+ plus calmodulin to purified synaptic vesicles resulted in a 4-fold increase in serine phosphorylation of synaptophysin, and this phosphorylation was antagonized by a peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II(. Purified rat forebrain CaM kinase II phosphorylated both purified synaptophysin and endogenous, vesicle-associated synaptophysin, and the resulting 2-dimensional chymotryptic phosphopeptide maps were similar to those derived from synaptophysin phosphorylated in cerebrocortical slices. These data demonstrate that Ca(2+)-dependent phosphorylation of synaptophysin, mediated by CaM kinase II, occurs under physiological conditions.
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PMID:Calcium-dependent serine phosphorylation of synaptophysin. 838 57

The Ca2+/calmodulin dependent protein kinase II (CaM kinase II) is thought to play an important part in glucose-stimulated insulin secretion. To determine which of the known subtypes (alpha, beta, gamma, delta) occur in insulin-secreting cells, we amplified all types of CaM kinase II by RT-PCR and found the beta3-, gamma-, delta2- and delta6-subtypes in RINm5F insulinoma cells. None of the other 8 delta-subtypes was present. Antibodies generated against the bacterially expressed association domain of the delta2-subtype recognized the recombinant gamma and delta-subtypes. In INS-1 and RINm5F cells, as well as freshly isolated rat islets, only a 55-kDa protein corresponding in size to the delta2-subtype expressed in NIH3T3 fibroblasts was detected. The delta2-subtype therefore appears to represent the predominant subtype of CaM kinase II present in insulin secreting cells. The enzyme was primarily associated with cytoskeletal structures, and very little was present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of its subcellular distribution was performed by sucrose and Nycodenz density gradient fractionation of INS-1 cells and detection of CaM kinase II delta by immune blots. The enzyme codistributed with insulin used as a marker for secretory granules but not with the lighter synaptic-like microvesicles detected with an antibody against synaptophysin, plasma membranes (syntaxin 1), lysosomes (arylsulfatase), or mitochondria (cytochrome c oxidase). CaM kinase II delta2 thus is identified as the subtype associated with insulin secretory granules and is likely to be involved in insulin secretion.
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PMID:Insulinoma cells contain an isoform of Ca2+/calmodulin-dependent protein kinase II delta associated with insulin secretion vesicles. 916 51

To investigate the physiological consequences of the increase in spine density induced by estradiol in pyramidal neurons of the hippocampus, we performed simultaneous whole cell recordings and Ca2+ imaging in CA1 neuron spines and dendrites in hippocampal slices. Four- to eight-days in vitro slice cultures were exposed to 17beta-estradiol (EST) for an additional 4- to 8-day period, and spine density was assessed by confocal microscopy of DiI-labeled CA1 pyramidal neurons. Spine density was doubled in both apical and basal dendrites of the CA1 region in EST-treated slices; consistently, a reduction in cell input resistance was observed in EST-treated CA1 neurons. Double immunofluorescence staining of presynaptic (synaptophysin) and postsynaptic (alpha-subunit of CaMKII) proteins showed an increase in synaptic density after EST treatment. The slopes of the input/output curves of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) postsynaptic currents were steeper in EST-treated CA1 neurons, consistent with the observed increase in synapse density. To characterize NMDA-dependent synaptic currents and dendritic Ca2+ transients during Schaffer collaterals stimulation, neurons were maintained at +40 mV in the presence of nimodipine, picrotoxin, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). No differences in resting spine or dendritic Ca2+ levels were observed between control and EST-treated CA1 neurons. Intracellular Ca2+ transients during afferent stimulation exhibited a faster slope and reached higher levels in spines than in adjacent dendrites. Peak Ca2+ levels were larger in both spines and dendrites of EST-treated CA1 neurons. Ca2+ gradients between spine heads and dendrites during afferent stimulation were also larger in EST-treated neurons. Both spine and dendritic Ca2+ transients during afferent stimulation were reversibly blocked by D, L-2-amino-5-phosphonovaleric acid (D,L-APV). The increase in spine density and the enhanced NMDA-dependent Ca2+ signals in spines and dendrites induced by EST may underlie a threshold reduction for induction of NMDA-dependent synaptic plasticity in the hippocampus.
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PMID:Estradiol increases spine density and NMDA-dependent Ca2+ transients in spines of CA1 pyramidal neurons from hippocampal slices. 1008 65

Ca2+/calmodulin-dependent protein kinase II is thought to participate in M3 muscarinic receptor-mediated acid secretion in gastric parietal cells. During acid secretion tubulovesicles carrying H+/K+-ATPase fuse with the apical membrane. We localized Ca2+/calmodulin-dependent protein kinase II from highly purified rabbit gastric tubulovesicles using Ca2+/calmodulin-dependent protein kinase II isoform-specific antibodies, in vitro phosphorylation and pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II activity by the potent Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. The presence of Ca2+/calmodulin-dependent protein kinase II in tubulovesicles was shown by immunoblot detection of both Ca2+/calmodulin-dependent protein kinase II-gamma (54 kDa) and Ca2+/calmodulin-dependent protein kinase II-delta (56.5 kDa). The immunoprecipitated Ca2+/calmodulin-dependent protein kinase II from tubulovesicles showed Ca2+/calmodulin-dependent protein kinase activity by phosphorylating autocamtide-II, a specific synthetic Ca2+/calmodulin-dependent protein kinase II substrate. KN-62 inhibited the in vitro autophosphorylation of tubulovesicle-associated Ca2+/calmodulin-dependent protein kinase II (IC50 = 11 nM). During the search for potential Ca2+/calmodulin-dependent protein kinase II substrates we identified different proteins associated with tubulovesicles, such as synaptophysin and beta-tubulin immunoreactivity, which were identified using specific antibodies. These targets are known to participate in intracellular membrane traffic. Ca2+/calmodulin-dependent protein kinase II is thought to play an important role in regulating tubulovesicular motor activity and therefore in acid secretion.
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PMID:Ca2+/calmodulin-dependent protein kinase II isoenzymes gamma and delta are both present in H+/K+-ATPase-containing rabbit gastric tubulovesicles. 1058 99

Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII alpha subunit. However, synaptophysin, SAP-102, and the GABA(A) receptor beta subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.
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PMID:Enrichment of N-methyl-D-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities. 1127 67

Epidemiological data indicate that low n-3 polyunsaturated fatty acids (PFA) intake is a readily manipulated dietary risk factor for Alzheimer's disease (AD). Studies in animals confirm the deleterious effect of n-3 PFA depletion on cognition and on dendritic scaffold proteins. Here, we show that in transgenic mice overexpressing the human AD gene APPswe (Tg2576), safflower oil-induced n-3 PFA deficiency caused a decrease in N-methyl-D-aspartate (NMDA) receptor subunits, NR2A and NR2B, in the cortex and hippocampus with no loss of the presynaptic markers, synaptophysin and synaptosomal-associated protein 25 (SNAP-25). n-3 PFA depletion also decreased the NR1 subunit in the hippocampus and Ca2+/calmodulin-dependent protein kinase (CaMKII) in the cortex of Tg2576 mice. These effects of dietary n-3 PFA deficiency were greatly amplified in Tg2576 mice compared to nontransgenic mice. Loss of the NR2B receptor subunit was not explained by changes in mRNA expression, but correlated with p85alpha phosphatidylinositol 3-kinase levels. Most interestingly, n-3 PFA deficiency dramatically increased levels of protein fragments, corresponding to caspase/calpain-cleaved fodrin and gelsolin in Tg2576 mice. This effect was minimal in nontransgenic mice suggesting that n-3 PFA depletion potentiated caspase activation in the Tg2576 mouse model of AD. Dietary supplementation with docosahexaenoic acid (DHA; 22 : 6n-3) partly protected from NMDA receptor subunit loss and accumulation of fodrin and gelsolin fragments but fully prevented CaMKII decrease. The marked effect of dietary n-3 PFA on NMDA receptors and caspase/calpain activation in the cortex of an animal model of AD provide new insights into how dietary essential fatty acids may influence cognition and AD risk.
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PMID:Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA receptors in the brain of a transgenic mouse model of Alzheimer's disease. 1610 43

Presynaptic proteins are readily identified by light microscopic immunocytochemistry, but immunodetection of postsynaptic proteins in brain sections proves difficult. We performed immunofluorescent double labeling for the NR1 subunit of the N-methyl-D-aspartate receptor (NMDAR) and the vesicular glutamate transporter 1 (VGLUT1). In material fixed with 4% paraformaldehyde, NMDAR staining in somatosensory cortex was restricted to the section surface, whereas presynaptic staining extended deeper into the tissue. Staining for postsynaptic proteins was enhanced in weakly fixed material and in tissue treated with pepsin, as previously reported, but tissue quality was impaired. Staining was also markedly enhanced, and without impairment of tissue quality, by treatment during perfusion with a mixture of inhibitors of proteases and the ubiquitin/proteosome system. We performed quantitative analysis of confocal images to study how immunostaining varies with depth into the tissue. Virtually all puncta immunopositive for VGLUT1 colocalized with synaptophysin puncta; these presynaptic puncta were most numerous 1-2 microm beneath the section surface. In contrast, puncta immunopositive for the NR1 subunit were most numerous at the surface, as were puncta immunopositive for the NR2 subunit, SynGAP, and CaMKII. Punctate staining for all postsynaptic proteins, but not presynaptic markers, was substantially enhanced in material pretreated with antiproteolytic agents. The large majority of NR1-positive puncta at the surface associated with VGLUT1 in this material are likely to represent synaptic contacts. Approximately eighty-five percent of VGLUT1-positive puncta in layers II-III of SI are associated with NR1-positive puncta, and approximately 80% are associated with NR2, SynGAP, and CaMKII. This approach may permit systematic analysis of the chemistry of glutamatergic synapses with light microscopic immunocytochemistry.
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PMID:Light microscopic identification and immunocytochemical characterization of glutamatergic synapses in brain sections. 1622 91

Frequent exposure of children to general anesthesia is common practice in modern medicine. Although previously unrecognized, recent in vitro and in vivo animal studies suggest that exposure to clinically relevant general anesthetics at the peak of brain development could be detrimental to immature mammalian neurons, as demonstrated by massive and widespread apoptotic neurodegeneration. The survival of the developing neurons presumably depends on proper and timely formation of synapses, for which synaptic proteins (e.g., synaptophysin, synaptobrevin, amphiphysin, synaptosomal-associated protein 25 [SNAP-25], and Ca(2+)/calmodulin-dependent protein kinase II [CaM kinase II]) are crucially important. Overinhibition of developing neurons impairs synaptic protein function and activity-induced synaptic plasticity, which could in turn result in permanent neuronal loss. To examine the effects of general anesthesia, the pharmacological agents known to cause extensive neuronal inhibition, on synaptic proteins, and neuronal survival at the peak of synaptogenesis, we exposed 7-day-old rat pups to general anesthesia (midazolam, 9 mg/kg of body weight, subcutaneously, followed by 6 h of nitrous oxide 75 vol% and isoflurane 0.75 vol%). We found that this general anesthesia causes permanent neuronal deletion in the most vulnerable brain regions-the cerebral cortex and the thalamus-while transiently modulating protein levels of synaptophysin, synaptobrevin, amphiphysin, SNAP-25, and CaM kinase II.
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PMID:Early exposure to general anesthesia causes significant neuronal deletion in the developing rat brain. 1807 65

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) belong to the family of perfluorinated compounds. They are used in industrial and consumer applications, e.g., clothing fabrics, carpets, and food packaging. PFOS and PFOA are present in the environment and are found in dust and human milk, which implies that newborns and toddlers can be directly exposed to these agents during brain development. Recently, we reported that PFOS and PFOA can cause neurobehavioral defects and changes in the cholinergic system, in the adult animal, when given directly to neonatal mice, and thereby showing similarities with other investigated persistent organic pollutants, such as dichloro-diphenyl-trichloroethan, polychlorinated biphenyls, and polybrominated diphenyl ethers (PBDEs). In recent studies, we have also seen that highly brominated PBDEs can affect the levels of proteins that are important for neuronal growth and synaptogenesis in the neonatal mouse brain. The present study shows that a single oral dose of either 21 micromol PFOS or PFOA/kg body weight (11.3 or 8.70 mg), given directly to the neonatal mice on postnatal day 10, significantly increased the levels of CaMKII, GAP-43, and synaptophysin in the hippocampus of the neonatal mouse. Both compounds significantly increased the levels of synaptophysin and tau in cerebral cortex, and PFOA also increased the levels of tau in hippocampus. These proteins are important for normal brain development, and altered levels of these proteins during a critical period of the brain growth spurts could be one of the mechanisms behind earlier reported behavioral defects.
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PMID:Neonatal exposure to PFOS and PFOA in mice results in changes in proteins which are important for neuronal growth and synaptogenesis in the developing brain. 1921 17

The cyclic AMP-dependent protein kinase (PKA) signaling pathway has been shown to be important in mechanisms of synaptic plasticity, although its direct and downstream signaling effects are not well understood. Using an in vitro model of eyeblink classical conditioning, we report that PKA has a critical role in initiating a signaling cascade that results in synaptic delivery of glutamate receptor 1 (GluR1)- and GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in abducens motor neurons during conditioning. PKA and the Ca(2+)-calmodulin-dependent protein kinases (CaMKs) II and IV are activated early in conditioning and are required for acquisition and expression of conditioned responses (CRs). cAMP-response-element-binding protein (CREB) is also activated early in conditioning but is blocked by coapplication of inhibitors to PKA and the CaMKs, suggesting that CREB is downstream of those signaling cascades. Moreover, evidence suggests that PKA activates extracellular signal-regulated kinase, which is also required for conditioning. Imaging studies after conditioning further indicate that colocalization of GluR1 AMPAR subunits with the synaptic marker synaptophysin requires PKA, but is insensitive to the N-methyl-d-aspartate receptor (NMDAR) inhibitor d,l-AP5. PKA activation also leads to synaptic localization of GluR4 subunits that, unlike GluR1, is dependent on NMDARs and is mediated by CaMKII. Together with previous studies, our findings support a two-stage model of AMPAR synaptic delivery during acquisition of classical conditioning. The first stage involves synaptic incorporation of GluR1-containing AMPARs that serves to activate silent synapses. This allows a second stage of NMDAR- and protein kinase C-dependent delivery of GluR4 AMPAR subunits that supports the acquisition of CRs.
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PMID:PKA has a critical role in synaptic delivery of GluR1- and GluR4-containing AMPARs during initial stages of acquisition of in vitro classical conditioning. 1926 6


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