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)

1. In visceral smooth muscles, both M(2) and M(3) muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCbeta activation. Thus, we studied their relevance for muscarinic cationic current (mI(CAT)) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca(2+)](i) (either using 50 microm EGTA or 50-100 microm fluo-3 for confocal fluorescence imaging) or with [Ca(2+)](i) 'clamped' at 100 nm using 10 mm BAPTA/CaCl(2) mixture. 2. Using a cAMP-elevating agent (1 microm isoproterenol) or a membrane-permeable cAMP analog (10 microm 8-Br-cAMP), we found no evidence for mI(CAT) modulation through a cAMP/PKA pathway. 3. With low [Ca(2+)](i) buffering, the PLC blocker U-73122 at 2.5 microm almost abolished mI(CAT), in some cases without any significant effect on [Ca(2+)](i). When [Ca(2+)](i) was buffered at 100 nm, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC(50)=0.5-0.6 microm) and shifted their activation curves positively. 4. U-73343, a weak PLC blocker, had no effect on GTPgammaS-induced mI(CAT), but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mm. Aristolochic acid and D-609, which inhibit PLA(2) and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI(CAT), suggesting that these enzymes were not involved. 5. InsP(3) (1 microm) in the pipette or OAG (20 microm) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca(2+) store depletion (evoked by InsP(3), or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI(CAT) in response to carbachol when [Ca(2+)](i) was strongly buffered to 100 nm. 6. It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca(2+) release, but also does so independently of InsP(3), DAG, Ca(2+) store depletion or a rise of [Ca(2+)](i). Our present results explain the previously established 'permissive' role of the M(3) receptor subtype in mI(CAT) generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.
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PMID:Phospholipase C, but not InsP3 or DAG, -dependent activation of the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells. 1466 35

Several receptors, including those for AVP (Arg8-vasopressin) and 5-HT (5-hydroxytryptamine), share an ability to stimulate PLC (phospholipase C) and so production of IP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol) in A7r5 vascular smooth muscle cells. Our previous analysis of the effects of AVP on Ca2+ entry [Moneer, Dyer and Taylor (2003) Biochem. J. 370, 439-448] showed that arachidonic acid released from DAG stimulated NO synthase. NO then stimulated an NCCE (non-capacitative Ca2+ entry) pathway, and, via cGMP and protein kinase G, it inhibited CCE (capacitative Ca2+ entry). This reciprocal regulation ensured that, in the presence of AVP, all Ca2+ entry occurred via NCCE to be followed by a transient activation of CCE only when AVP was removed [Moneer and Taylor (2002) Biochem. J. 362, 13-21]. We confirm that, in the presence of AVP, all Ca2+ entry occurs via NCCE, but 5-HT, despite activating PLC and evoking release of Ca2+ from intracellular stores, stimulates Ca2+ entry only via CCE. We conclude that two PLC-coupled receptors differentially regulate CCE and NCCE. We also address evidence that, in some A7r5 cells lines, AVP fails either to stimulate NCCE or inhibit CCE [Brueggemann, Markun, Barakat, Chen and Byron (2005) Biochem. J. 388, 237-244]. Quantitative PCR analysis suggests that these cells predominantly express TRPC1 (transient receptor potential canonical 1), whereas cells in which AVP reciprocally regulates CCE and NCCE express a greater variety of TRPC subtypes (TRPC1=6>2>3).
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PMID:Different phospholipase-C-coupled receptors differentially regulate capacitative and non-capacitative Ca2+ entry in A7r5 cells. 1591 94

Mechanical ventilation is a valuable treatment regimen for respiratory failure. However, mechanical ventilation (especially with high tidal volumes) is implicated in the initiation and/or exacerbation of lung injury. Hence, it is important to understand how the cells that line the inner surface of the lung [alveolar epithelial cells (AECs)] sense cyclic stretching. Here, we tested the hypothesis that matrix molecules, via their interaction with surface receptors, transduce mechanical signals in AECs. We first determined that rat AECs secrete an extracellular matrix (ECM) rich in anastamosing fibers composed of the alpha3 laminin subunit, complexed with beta1 and gamma1 laminin subunits (i.e. laminin-6), and perlecan by a combination of immunofluorescence microscopy and immunoblotting analyses. The fibrous network exhibits isotropic expansion when exposed to cyclic stretching (30 cycles per minute, 10% strain). Moreover, this same stretching regimen activates mitogen-activated-protein kinase (MAPK) in AECs. Stretch-induced MAPK activation is not inhibited in AECs treated with antagonists to alpha3 or beta1 integrin. However, MAPK activation is significantly reduced in cells treated with function-inhibiting antibodies against the alpha3 laminin subunit and dystroglycan, and when dystroglycan is knocked down in AECs using short hairpin RNA. In summary, our results support a novel mechanism by which laminin-6, via interaction with dystroglycan, transduces a mechanical signal initiated by stretching that subsequently activates the MAPK pathway in rat AECs. These results are the first to indicate a function for laminin-6. They also provide novel insight into the role of the pericellular environment in dictating the response of epithelial cells to mechanical stimulation and have broad implications for the pathophysiology of lung injury.
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PMID:Laminin-6 assembles into multimolecular fibrillar complexes with perlecan and participates in mechanical-signal transduction via a dystroglycan-dependent, integrin-independent mechanism. 1592 48

The effects of (+/-)3,4-methylenedioxyamphetamine (MDA) were studied in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage-clamp method. The RP4 neuron generated spontaneous action potentials. Extracellular or intracellular application of MDA elicited action potential bursts of the central RP4 neuron. The action potential bursts elicited by MDA were not blocked when neurons were immersed in high-Mg2+ solution, Ca2+-free solution, nor after continuous perfusion with atropine, d-tubocurarine, propranolol, prazosin, haloperidol, sulpiride or methiothepin. Notably, the induction of action potential bursts was blocked by pretreatment with protein kinase C (PKC) inhibitors, chelerythrine and Ro 31-8220, but not by protein kinase A (PKA) inhibitors, KT-5720 and H89, nor by the phospholipase C (PLC) inhibitor, U73122. PKC activators, i.e., phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acety-sn-glycerol (OAG; a membrane-permeant DAG analog), facilitate the induction of action potential bursts elicited by MDA. Voltage-clamp studies revealed that MDA decreased the delayed rectifying K+ current (I(KD)) of the RP4 neuron. Further, although Ro 31-8220 did not affect the I(KD), Ro 31-8220 decreased the inhibitory effect of MDA on the I(KD). These results suggest that the generation of action potential bursts elicited by MDA was not due to (1) the synaptic effects of neurotransmitters, (2) the cholinergic, adrenergic, dopaminergic or serotoninergic receptors of the excitable membrane. Instead, the MDA-elicited action potential bursts are closely related to PKC activity and the inhibitory effects on the I(KD).
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PMID:(+/-)3,4-Methylenedioxyamphetamine elicits action potential bursts in a central snail neuron. 1715 97

The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCeta, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCbeta3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production.
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PMID:Phospholipase C beta3 is a key component in the Gbetagamma/PKCeta/PKD-mediated regulation of trans-Golgi network to plasma membrane transport. 1749 41

Beneficial effects of low-concentration chemical stressors have been investigated previously in different model systems. The symptoms of stimulation are known from earlier studies, but information about the mechanism is at an initial stage. In the present work, the mechanism of stimulation of low-concentration Cd (5 x 10(-8)M) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, 10(-7)M) was investigated in barley seedlings. In treated plants, the amount of cytokinins increased in roots and, after being transported to the leaves, they caused stimulation there. To identify the signal transduction pathway(s) involved in the primary stimulation of cytokinin synthesis (and/or activation) in roots, specific phosphatidylinositol-4,5-bisphosphate-inositol-1,4,5-triphosphate/diacylglycerol (PIP(2)-IP(3)/DAG) and mitogen activated protein kinase (MAPK) signaling pathway inhibitors were added to the nutrient solution, and all proved to be effective, eliminating the stimulation by the stressors. Measurements of superoxide dismutase (SOD, EC 1.15.1.1) activity and the amount of malonyldialdehyde (MDA) showed that the increased amount of Cd did not cause oxidative stress in the roots, and no oxidative stress was found in the leaves, where Cd did not even accumulate. DCMU slightly increased the activity of SOD after 1 week in roots, but did not cause lipid peroxidation. In leaves, there was no oxidative stress upon treatment with DCMU. Thus, oxidative stress cannot be responsible for the stimulation with low-concentration stressors, as they changed the activity of SOD differently, while being equally stimulative for the plants.
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PMID:Investigation into the mechanism of stimulation by low-concentration stressors in barley seedlings. 1843 Apr 89

Signals transmitted via multichain immunoreceptors control the development, differentiation and activation of hematopoetic cells. The cytoplasmic parts of these receptors contain immunoreceptor tyrosine-based activation motifs (ITAMs) that upon phosphorylation by members of the Src tyrosine kinase family orchestrate a complex set of signaling events involving tyrosine phosphorylation, generation of second messengers like DAG, IP3 and Ca2+, activation of effector molecules like Ras and MAPKs and the translocation and activation of transcription factors like NFAT, API and NF-kB. Spatial and temporal organization of these signaling events is essential both to connect the receptors to downstream cascades as well as to control the functional outcome of the immune activation. Throughout this process control and fine-tuning of the different signals are necessary both for effective immune function and in order to avoid inappropriate or exaggerated immune activation and autoimmunity. This control includes modulating mechanisms that set the threshold for activation and reset the activation status after an immune response has been launched. One immunomodulating pathway is the cAMP-protein kinase A-Csk pathway scaffolded by a supramolecular complex residing in lipid rafts with the A kinase-anchoring protein (AKAP) ezrin, the Csk-binding protein PAG and a linker between the two, EBP50. Failure of correct scaffolding and loss of spatiotemporal control can potentially have severe consequences, leading to immune failure or autoimmunity. The clinical relevance of supramolecular complexes specifically organized by scaffolding proteins in regulating immune activity and the specter of genetic diseases linked to different signaling components suggest that protein-protein contact surfaces can be potential targets for drug intervention. It is also of interest to note that different pathogens have evolved strategies to specifically modulate signal integration, thereby rewiring the signal in a way beneficial for their survival. In addition to demonstrating the importance of different signal processes, these adaptations are elegant illustrations of the potential for drug targeting of protein assembly. This chapter reviews some of the important scaffolding events downstream of immunoreceptors with focus on signaling transduction through the T-cell receptor (TCR).
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PMID:Molecular architecture of signal complexes regulating immune cell function. 1849 Oct 59

Saturated fatty acids, such as palmitate, promote accumulation of ceramide, which impairs activation and signalling of PKB (protein kinase B; also known as Akt) to important end points such as glucose transport. SPT (serine palmitoyl transferase) is a key enzyme regulating ceramide synthesis from palmitate and represents a potential molecular target in curbing lipid-induced insulin resistance. In the present study we explore the effects of palmitate upon insulin action in L6 muscle cells in which SPT expression/activity has been decreased by shRNA (small-hairpin RNA) or sustained incubation with myriocin, an SPT inhibitor. Incubation of L6 myotubes with palmitate (for 16 h) increases intramyocellular ceramide and reduces insulin-stimulated PKB activation and glucose uptake. PKB inhibition was not associated with impaired IRS (insulin receptor substrate) signalling and was ameliorated by short-term treatment with myriocin. Silencing SPT expression (approximately 90%) by shRNA or chronic cell incubation with myriocin (for 7 days) markedly suppressed SPT activity and palmitate-driven ceramide synthesis; however, challenging these muscle cells with palmitate still inhibited the hormonal activation of PKB. This inhibition was associated with reduced IRS1/p85-PI3K (phosphoinositide 3-kinase) coupling that arises from diverting palmitate towards greater DAG (diacylglycerol) synthesis, which elevates IRS1 serine phosphorylation via activation of DAG-sensitive PKCs (protein kinase Cs). Treatment of SPT-shRNA cells or those treated chronically with myriocin with PKC inhibitors antagonized palmitate-induced loss in insulin signalling. The findings of the present study indicate that SPT plays a crucial role in desensitizing muscle cells to insulin in response to incubation with palmitate. While short-term inhibition of SPT ameliorates palmitate/ceramide-induced insulin resistance, sustained loss/reduction in SPT expression/activity promotes greater partitioning of palmitate towards DAG synthesis, which impacts negatively upon IRS1-directed insulin signalling.
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PMID:Modulating serine palmitoyl transferase (SPT) expression and activity unveils a crucial role in lipid-induced insulin resistance in rat skeletal muscle cells. 1892 31

Previously, we showed that laminin-binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G-protein (Galphabetagamma) to bind, changing the activation state of the Gsalpha subunit. Others have shown that laminin-binding to the DGC also leads to Akt activation. Gbetagamma, released when Gsalpha is activated, is known to bind phosphatidylinositol-3-kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from Gbetagamma, using immunoprecipitation and immunoblotting, and purified Gbetagamma. In the presence of laminin, PI3K-binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin-binding to alpha-dystroglycan, prevent PI3K-binding to the DGC. Purified bovine brain Gbetagamma also caused PI3K and Akt activation. These results show that DGC-Gbetagamma is binding PI3K and activating pAkt in a laminin-dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin beta1 compared to normal muscle. This integrin binds laminin, Gbetagamma, and PI3K. Collectively, these suggest that PI3K is an important target for the Gbetagamma, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis-regulation of the laminin-DGC-Gbetagamma-PI3K-Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Upregulating integrin beta1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease.
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PMID:Dystrophin glycoprotein complex-associated Gbetagamma subunits activate phosphatidylinositol-3-kinase/Akt signaling in skeletal muscle in a laminin-dependent manner. 1911 13

The AMP-stimulated protein kinase regulates epithelial polarity under conditions of energy depletion by phosphorylating the myosin regulatory light chain. In this issue of Developmental Cell, Mirouse et al. demonstrate that dystroglycan and perlecan, an extracellular matrix receptor and its ligand, help localize myosin regulatory light chain, making it available for phosphorylation by AMP kinase in response to energy stress.
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PMID:Dystroglycan and AMP kinase: polarity's protectors when the power goes out. 1915 20


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