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)

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.
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PMID:Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo. 1548 25

Ca(v)2.1 and Ca(v)2.2 channels conduct P/Q-type and N-type Ca(2+) currents that initiate neurotransmission and bind SNARE proteins through a synaptic protein interaction (synprint) site. PKC and CaMKII phosphorylate the synprint site and inhibit SNARE protein binding in vitro. Here we identify two separate microdomains that each bind syntaxin 1A and SNAP-25 in vitro and are regulated by PKC phosphorylation at serines 774 and 898 and CaMKII phosphorylation at serines 784 and 896. Activation of PKC resulted in its recruitment to and phosphorylation of Ca(V)2.2 channels, but PKC phosphorylation did not dissociate Ca(V)2.2 channel/syntaxin 1A complexes. Chimeric Ca(V)2.1a channels containing the synprint site of Ca(v)2.2 gain modulation by syntaxin 1A, which is blocked by PKC phosphorylation at the sites identified above. Our results support a bipartite model for the synprint site in which each SNARE-binding microdomain is controlled by a separate PKC and CaMKII phosphorylation site that regulates channel modulation by SNARE proteins.
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PMID:Mechanism of SNARE protein binding and regulation of Cav2 channels by phosphorylation of the synaptic protein interaction site. 1560 37

Synaptosomal-associated protein of 25kDa (SNAP-25), a member of the SNARE proteins essential for neurotransmitter release, is phosphorylated at Ser(187) in PC12 cells and in the rat brain in a protein kinase C-dependent manner. It remains unclear how the phosphorylation of SNAP-25 is regulated during development and by neuronal activity. We studied the mode of SNAP-25 phosphorylation at Ser(187) in the rat brain using an anti-phosphorylated SNAP-25 antibody. Both the expression and phosphorylation of SNAP-25 increased remarkably during the early postnatal period, but their onsets were quite different. SNAP-25 expression was detected as early as embryonic Day 18, whereas the phosphorylation of SNAP-25 could not be detected until postnatal Day 4. A delay in the onset of phosphorylation was also observed in cultured rat hippocampal neurons. The phosphorylation of SNAP-25 was regulated in a neuronal activity-dependent manner and, in the rat hippocampus, decreased by introducing seizures with kainic acid. These results clearly indicated that the phosphorylation of SNAP-25 at Ser(187) is regulated in development- and neuronal activity-dependent manners, and is likely to play important roles in higher brain functions.
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PMID:Development- and activity-dependent regulation of SNAP-25 phosphorylation in rat brain. 1697 78

The norepinephrine (NE) transporter (NET) terminates noradrenergic signaling by clearing released NE at synapses. The activity of NET can be rapidly regulated by depolarization and receptor activation via Ca2+ and kinase/phosphatase-linked pathways. The SNARE protein syntaxin 1A (SYN1A) interacts with NET and influences transporter surface trafficking and catalytic activity. In this study, we establish a link between changes in intracellular Ca2+ and SYN1A/NET interactions. SYN1A influenced NE transport only in the presence of Ca2+ in brain cortical synaptosomes. Although NET/SYN1A associations were sensitive to manipulations of Ca2+ in CHO cells, in vitro binding experiments using purified NET and SYN1A fusion proteins demonstrated a lack of direct Ca2+ sensitivity. Disruption of NET/SYN1A interaction abolished inhibition of NE transport by phorbol ester (PMA) to activate protein kinase C (PKC), but had no effect on transport inhibition by the Ca2+ calmodulin kinase (CaMK) inhibitor KN93. Furthermore, PMA enhanced Ca2+-dependent modulation of NE transport in synaptosomes. Our data reveal roles for SYN1A in the Ca2+-dependent regulation of NET, likely reliant on regulation by PKC signaling, but independent of CaMK.
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PMID:Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A. 1718 89

The vesicle priming protein Munc13-1 is regulated by diacylglycerol (DAG) and is therefore hypothesized to play a role in the control of neurotransmitter release by phospholipase C (PLC)-coupled receptors. We combined voltage-clamp recordings of voltage-gated Ca2+ channels (VGCCs) and high-resolution capacitance measurements to investigate the mechanism of receptor-mediated modulation of exocytosis in bovine chromaffin cells. Activation of endogenous H1 G(q)-protein-coupled receptors (G(q)PCRs) by histamine potentiated stimulus-coupled secretion despite concurrently inhibiting Ca2+ influx through VGCCs. Histamine increased the size of the readily releasable pool of vesicles and in particular a subpool of fusion-competent vesicles localized in close proximity to VGCCs. Pharmacological characterization showed that potentiation of exocytosis depended on the activation of PLC but not protein kinase C. Overexpression of wild-type Munc13-1 by adenoviral infection had no effect on histamine-induced potentiation per se, whereas DAG-insensitive Munc13-1(H567K) completely abolished it. This is the first endogenous mammalian G(q)PCR signaling pathway identified that engages Munc13-1 to increase stimulus-coupled secretion by recruiting vesicles to the immediately releasable pool. G(q)PCRs are therefore able to control exocytosis at the level of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex formation to produce rapid, short-term potentiation of the secretory output of neurons and endocrine cells.
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PMID:Potentiation of exocytosis by phospholipase C-coupled G-protein-coupled receptors requires the priming protein Munc13-1. 1720 88

The pancreatic acinus is the functional unit of the exocrine pancreas whose role is to secrete zymogens into the gut lumen for food digestion via apical exocytosis. We previously reported that supramaximal CCK induced apical blockade and redirected exocytosis to ectopic sites on the basolateral plasma membrane (BPM) of this polarized cell, leading to pancreatitis. Basolateral exocytosis was mediated by protein kinase C phosphorylation of BPM Munc18c, causing its displacement into the cytosol and activation of BPM-bound Syntaxin-4 to form a SNARE complex. To mimic the conditions of alcoholic pancreatitis, we now examined whether 20 mm alcohol followed by submaximal CCK might mimic supramaximal CCK in inducing these pathologic exocytotic events. We show that a non-secretory but clinically relevant alcohol concentration (20 mm) inhibited submaximal CCK (50 pM)-stimulated amylase secretion by blocking apical exocytosis and redirecting exocytosis to less efficient BPM, indeed mimicking supramaximal CCK (10 nM) stimulation. We further demonstrate that basolateral exocytosis caused by both stimulation protocols is mediated by PKC alpha-induced phosphorylation of Munc18c: 1) PKC alpha is activated, which binds and induces phosphorylation of PM-Munc18c at a Thr site, and these events can be inhibited by PKC alpha blockade; 2) PKC alpha inhibition blocks Munc18c displacement from the BPM; 3) PKC alpha inhibition prevents basolateral exocytosis but does not rescue apical exocytosis. We conclude that 20 mm alcohol/submaximal CCK as well supramaximal CCK stimulation can trigger pathologic basolateral exocytosis in pancreatic acinar cells via PKC alpha-mediated activation of Munc18c, which enables Syntaxin-4 to become receptive in forming a SNARE complex in the BPM; and we further postulate this to be an underlying mechanism contributing to alcoholic pancreatitis.
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PMID:Alcohol/cholecystokinin-evoked pancreatic acinar basolateral exocytosis is mediated by protein kinase C alpha phosphorylation of Munc18c. 1732 28

The phosphorylation targets that mediate the enhancement of exocytosis by PKC are unknown. PKC phosporylates the SNARE protein SNAP-25 at Ser-187. We expressed mutants of SNAP-25 using the Semliki Forest Virus system in bovine adrenal chromaffin cells and then directly measured the Ca2+ dependence of exocytosis using photorelease of caged Ca2+ together with patch-clamp capacitance measurements. A flash of UV light used to elevate [Ca2+](i) to several microM and release the highly Ca2+-sensitive pool (HCSP) of vesicles was followed by a train of depolarizing pulses to elicit exocytosis from the less Ca2+-sensitive readily releasable pool (RRP) of vesicles. Carbon fiber amperometry confirmed that the amount and kinetics of catecholamine release from individual granules were similar for the two phases of exocytosis. Mimicking PKC phosphorylation with expression of the S187E SNAP-25 mutant resulted in an approximately threefold increase in the HCSP, whereas the response to depolarization increased only 1.5-fold. The phosphomimetic S187D mutation resulted in an approximately 1.5-fold increase in the HCSP but a 30% smaller response to depolarization. In vitro binding assays with recombinant SNARE proteins were performed to examine shifts in protein-protein binding that may promote the highly Ca2+-sensitive state. The S187E mutant exhibited increased binding to syntaxin but decreased Ca2+-independent binding to synaptotagmin I. Mimicking phosphorylation of the putative PKA phosphorylation site of SNAP-25 with the T138E mutation decreased binding to both syntaxin and synaptotagmin I in vitro. Expressing the T138E/ S187E double mutant in chromaffin cells demonstrated that enhancing the size of the HCSP correlates with an increase in SNAP-25 binding to syntaxin in vitro, but not with Ca2+-independent binding of SNAP-25 to synaptotagmin I. Our results support the hypothesis that exocytosis triggered by lower Ca2+ concentrations (from the HCSP) occurs by different molecular mechanisms than exocytosis triggered by higher Ca2+ levels.
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PMID:Phosphomimetic mutation of Ser-187 of SNAP-25 increases both syntaxin binding and highly Ca2+-sensitive exocytosis. 1732 94

Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for 5 days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by Western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by Western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-18/syntaxin 1A was decreased. Serine phosphorylation of SNAP-25, syntaxin 1A, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactin-treated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22 mM glucose, 40 mM K(+), 200 microM carbachol and 1 microM PMA. The prolactin-induced increase in the formation of SNARE complex and syntaxin 1A phosphorylation was inhibited by PD098059 and U0126, inhibitors of the MAPK pathway. These findings indicate that prolactin primes pancreatic beta-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery and the MAPK/PKC pathway is important for this effect.
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PMID:Prolactin modulates the association and phosphorylation of SNARE and kinesin/MAP-2 proteins in neonatal pancreatic rat islets. 1757 85

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Regulated secretion requires SNARE proteins and, in neurons, also synaptotagmin I and complexin. Recent reports suggest that complexin imposes a fusion block that is released by Ca(2+) and synaptotagmin I. However, no direct evidence for this model in secreting cells has been provided and whether this complexin/synaptotagmin interplay functions in other types of secretion is unknown. In this report, we show that the C2B domain of synaptotagmin VI and an anti-complexin antibody blocked the formation of trans SNARE complexes in permeabilized human sperm, and that this effect was reversed by adding complexin. In contrast, an excess of complexin stopped exocytosis at a later step, when SNAREs were assembled in loose trans complexes. Interestingly, this blockage was released by the addition of the synaptotagmin VI C2B domain in the presence of Ca(2+). We have previously demonstrated that the activity of this domain is regulated by protein kinase C-mediated phosphorylation. Here, we show that a phosphomimetic mutation in the polybasic region of the C2B domain strongly affects its Ca(2+) and phospholipids binding properties. Importantly, this mutation completely abrogates its ability to rescue the complexin block. Our results show that the functional interplay between complexin and synaptotagmin has a central role in a physiological secretion event, and that this interplay can be modulated by phosphorylation of the C2B domain.
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PMID:Complexin/synaptotagmin interplay controls acrosomal exocytosis. 1761 20

Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell (RBC) marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP2/RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin (PNA) established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in the same subcellular compartment of the horizontal cell. Taken together, the strong expression of these two SNARE proteins in the processes and endings of horizontal cells at rod and cone terminals suggests that horizontal cell axons and dendrites are likely sites of exocytotic activity.
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PMID:Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes. 1764 Apr 43


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