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)

The mechanisms of activation of cytoplasmic phospholipase A2 (cPLA2) are complex and incompletely defined. In Chinese hamster ovary (CHO) cells, receptor stimulation of cPLA2 is due to the interaction of pathways involving the alpha subunits of at least two guanine-nucleotide-binding (G) proteins, G alpha i2 and G alpha q. Activation of cPLA2 is inhibited by pertussis toxin and G alpha i2 mutants. In addition, activation of phospholipase C via G alpha q results in increased intracellular calcium ([Ca2+]i) and activation of protein kinase C, both of which interact with and activate cPLA2. The present study was undertaken to analyze the mechanism of interaction of G alpha i2 with the phospholipase-C-stimulated pathway in the activation of cPLA2. We addressed this question using a dominant negative G alpha i2 mutant, [G203T]G alpha i2, in which Gly203 is mutated to Thr. [G203T]G alpha i2 inhibits ATP receptor activation of cPLA2. The effect of [G203T]G alpha i2 was specific to G alpha i2-activated pathways, as shown by its lack of effect on other purinergic receptor stimulated pathways: ATP stimulation of [Ca2+]i or mitogen-activated protein kinase phosphorylation is unaltered by [G203T]G alpha i2. We addressed the possibility that the activation of cPLA2 by Ca2+ and/or protein kinase C is dependent on G alpha i2. Activation of cPLA2 by the Ca2+ ionophore, ionomycin, was inhibited by 61 +/- 9% (n = 5) in [G203T]G alpha i2-expressing cells; however the ionomycin-induced [Ca2+]i rise was unaffected by [G203T]G alpha i2. Thus, [G203T]G alpha i2. specifically inhibits Ca2+ activation of cPLA2. In contrast, activation of cPLA2 via protein kinase C by phorbol 12-myristate 13-acetate was unaffected by [G203T]G alpha i2. Our results demonstrate that Ca2+ but not phorbol ester activation of cPLA2 in CHO cells is G alpha i2-dependent. The possibility is discussed that G alpha i2 is downstream of Ca2+ but upstream of protein kinase C activation of cPLA2.
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PMID:The guanine-nucleotide-binding protein subunit G alpha i2 is involved in calcium activation of phospholipase A2. Effects of the dominant negative G alpha i2 mutant, [G203T]G alpha i2, on activation of phospholipase A2 in Chinese hamster ovary cells. 760 Oct 96

Constitutively activated mutants of the G alpha 12 class of G proteins, G alpha 12(Q229L) and G alpha 13(Q226L), were transiently expressed in COS-1 cells, and the activity of amiloride-sensitive Na+/H+ exchanger was measured. The expression of either G alpha 12(Q229L) or G alpha 13(Q226L) increased the basal activity of the amiloride-sensitive exchanger by 2-5-fold. Regulation of this activation by other G protein signaling pathways was investigated by the transient expression of constitutively activated G protein mutants of G alpha s(Q227L), G alpha i2(Q205L), and G alpha q(Q209L) in COS-1 cells. Only G alpha q showed a similar activation of the exchanger. Chronic treatment of the transfected cells with 4 beta-phorbol 12-myristate 13-acetate to deplete the endogenous protein kinase C completely inhibited the activation of the antiporter by G alpha 12(Q229L), whereas activation by G alpha 13(Q226L) remained unaffected. These results indicated that both G alpha 12 and G alpha 13 can activate Na+/H+ exchanger by two distinct signaling pathways. G alpha 12 activation of the exchanger was dependent on protein kinase C pathway, whereas G alpha 13 activation was not. These studies define the involvement of G alpha 12 class of G proteins, for which no function has been assigned yet, in the activation of Na+/H+ exchanger.
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PMID:Protein kinase C-dependent and -independent activation of Na+/H+ exchanger by G alpha 12 class of G proteins. 816 78

Recent work indicates that the therapeutic action of lithium may be mediated through perturbation of postreceptor second messenger systems. To elucidate further the postreceptor cellular sites of action(s) of lithium, the effect of chronic lithium treatment on various components of the receptor-activated phosphoinositide pathway was investigated. We found that chronic administration of lithium (0.2% LiCl, 21 days) to adult male rats did not significantly affect phosphoinositide hydrolysis in cerebral cortical slices induced by carbachol (1 mM) or NaF (10 mM). Nor did the same treatment alter the carbachol (1 mM) potentiation of guanosine 5'-(gamma-thio)triphosphate (30 microM) stimulation of phosphoinositide hydrolysis (an index of receptor/G protein coupling) in cortical membranes. Immunoblotting studies revealed no changes in the levels of G alpha q/11 immunoreactivity in the cortex after chronic lithium treatment. The levels of protein kinase C, as revealed by specific binding of [3H]phorbol dibutyrate ([3H]PDBu), were significantly reduced in the cytosolic fraction and increased in the particulate fraction of rat cortex after chronic lithium, whereas the KD of [3H]PDBu binding remained relatively constant. A small and insignificant decrease in the density of [3H]inositol 1,4,5-trisphosphate binding was also found in the cortex. The above data suggest that chronic lithium treatment affects neither the muscarinic cholinergic-linked phosphoinositide turnover nor the putative G protein alpha subunit (G alpha q/11) responsible for phospholipase C activation. However, a possible translocation and activation of protein kinase C activity may be significant in the therapeutic effect of this mood-stabilizing agent.
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PMID:Lithium modulation of phosphoinositide signaling system in rat cortex: selective effect on phorbol ester binding. 822 88

Using the yeast two-hybrid system we have identified a human protein, GAIP (G Alpha Interacting Protein), that specifically interacts with the heterotrimeric GTP-binding protein G alpha i3. Interaction was verified by specific binding of in vitro-translated G alpha i3 with a GAIP-glutathione S-transferase fusion protein. GAIP is a small protein (217 amino acids, 24 kDa) that contains two potential phosphorylation sites for protein kinase C and seven for casein kinase 2. GAIP shows high homology to two previously identified human proteins, GOS8 and 1R20, two Caenorhabditis elegans proteins, CO5B5.7 and C29H12.3, and the FLBA gene product in Aspergillus nidulans--all of unknown function. Significant homology was also found to the SST2 gene product in Saccharomyces cerevisiae that is known to interact with a yeast G alpha subunit (Gpa1). A highly conserved core domain of 125 amino acids characterizes this family of proteins. Analysis of deletion mutants demonstrated that the core domain is the site of GAIP's interaction with G alpha i3. GAIP is likely to be an early inducible phosphoprotein, as its cDNA contains the TTTTGT sequence characteristic of early response genes in its 3'-untranslated region. By Northern analysis GAIP's 1.6-kb mRNA is most abundant in lung, heart, placenta, and liver and is very low in brain, skeletal muscle, pancreas, and kidney. GAIP appears to interact exclusively with G alpha i3, as it did not interact with G alpha i2 and G alpha q. The fact that GAIP and Sst2 interact with G alpha subunits and share a common domain suggests that other members of the GAIP family also interact with G alpha subunits through the 125-amino-acid core domain.
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PMID:GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain. 852 74

The connection between agonist-induced desensitization and down-regulation of 5-hydroxytryptamine2A (5-HT2A) receptors was examined in a clonal cell line that stably expresses the 5-HT2A receptor. Brief (2-hr) and prolonged (24-hr) exposure to the agonist quipazine or the agonist 4-iodo-(2,5-dimethoxy)- phenylisopropylamine (DOI) diminished 5-HT2A receptor-mediated phosphoinositide hydrolysis; no change in 5-HT2A receptor number or affinity was measured after 24 hr of exposure to DOI or quipazine. Immunohistochemical studies demonstrated that a 24-hr exposure to DOI did not alter surface 5-HT2A receptor immunoreactivity. Western blot analysis with G alpha q- and G alpha 11-selective antibodies indicate that a 24-hr agonist exposure did not alter the levels of phospholipase C-dependent G proteins. These results suggest that desensitization after prolonged DOI exposure can occur via a process independent of the levels of phospholipase C-coupled G proteins. Studies with a mutant 5-HT2A receptor (F340L) indicated that binding per se is not sufficient for desensitization. Down-regulation of the protein kinase C isozymes alpha and epsilon by overnight exposure to phorbol-12,13-dibutyrate attenuated the intermediate phase (i.e., after 2-6 hr of agonist exposure) of DOI- and quipazine-induced desensitization. These results indicate that the intermediate phase of DOI-induced desensitization is mediated by the alpha- and/or epsilon-protein kinase C isozymes but that neither is involved in the later phase (i.e., after 24 hr of agonist exposure) of desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:5-Hydroxytryptamine2A (5-HT2A) receptor desensitization can occur without down-regulation. 853 Nov 39

Recent studies have shown that G proteins are a potential regulatory site in the transmembrane signaling cascade. The aim of this study was to examine the effects of prolonged agonist exposure on expression of the Gq class of G protein alpha subunits (G alpha q/G alpha 11) in cultured rat vascular smooth muscle cells (VSMC). Treatment with 100 nM angiotensin II (Ang II) led to a substantial sustained down-regulation of cellular levels of immunologically detectable G alpha q/G alpha 11 by 50% within 6 hr. The effect of Ang II was dose dependent with an EC50 of 2 nM and was specifically blocked by the vascular type-1 Ang II receptor-specific antagonist losartan. The Ang II-induced reduction in cellular levels of G protein alpha subunits was specific for G alpha q/G alpha 11. The calcium ionophore ionomycin or activators of ubiquitous protein kinases (phorbol-12-myristate-13-acetate, forskolin, and 8-bromo-cGMP) did not mimic the effects of Ang II. However, [Arg8]vasopressin also induced a significant loss in cellular G alpha q/G alpha 11 levels. Ang II-induced G alpha q/G alpha 11 down-regulation was reversed by prevention of cellular receptor processing with phenylarsine oxide or chronic potassium depletion. The effects of Ang II on G alpha q/G alpha 11 levels were inhibited when protein kinase C activity was abolished. G alpha q mRNA levels were down-regulated by 30% after 4-hr incubation with Ang II, in part by transcriptional regulation. Although a short term vasopressin pretreatment had no effect on inositol-1,4,5-trisphosphate (IP3) generation in response to subsequent Ang II stimulation, a partial heterologous desensitization of the IP3 response was induced after a long term vasopressin pretreatment, which concurrently down-regulated cellular G alpha q/G alpha 11 levels. Homologous desensitization of IP3 generation on a second Ang II stimulation was observed after both a short and long term Ang II pretreatment. In conclusion, prolonged exposure to Ang II induces down-regulation of cellular G alpha q/G alpha 11 levels in intact VSMC. The effect of Ang II appears to be mediated by the signaling pathway sensitive to inhibition of receptor processing. The present study raises the possibility that agonist-induced G alpha q/G alpha 11 down-regulation participates in the mechanism of long term desensitization of the G alpha q/G alpha 11-mediated signaling system in VSMC.
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PMID:Prolonged exposure to agonist results in a reduction in the levels of the Gq/G11 alpha subunits in cultured vascular smooth muscle cells. 856 18

Parafollicular (PF) cells secrete 5-hydroxytryptamine in response to increased extracellular Ca2+ ([Ca2+]e). This stimulus causes Cl- channels in PF secretory vesicles to open, leading to vesicle acidification. PF cells express a plasmalemmal heptahelical receptor (CaR) that binds Ca2+, Gd3+, and Ba2+. We now report that the CaR mediates vesicle acidification. Ca2+, Gd3+, and Ba2+ induced vesicle acidification, which was independent of channel-mediated Ca2+ entry. Agonist-induced vesicle acidification was blocked by pertussis toxin, inhibitors of phosphatidylinositol-phospholipase C, calmodulin, NO synthase, guanylyl cyclase, or protein kinase G. PF cells contained NO synthase immunoreactivity, and vesicles were acidified by NO donors and dibutyryl cGMP. [Ca2+]e, and Gd3+ mobilized thapsigargin-sensitive internal Ca2+ stores. [35S]G alpha i and [35S]G alpha q were immunoprecipitated from PF membranes incubated with agonists in the presence of [35S]adenosine 5'-O-(thiotriphosphate). Labeling of G alpha i but not G alpha q was antagonized by pertussis toxin. Vesicles acidified in response to activation of protein kinase C; however, protein kinase C inhibition blocked calcium channel- but not CaR-dependent acidification. We propose the following signal transduction pathway: CaR -> Gi -> phosphatidylinositol-phospholipase C -> inositol 1,4,5-trisphosphate -> [Ca2+]i -> Ca2+/calmodulin -> NO synthase -> NO -> guanylyl cyclase -> cGMP -> protein kinase G -> opens vesicular Cl- channel.
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PMID:Acidification of serotonin-containing secretory vesicles induced by a plasma membrane calcium receptor. 862 45

The heterotrimeric G proteins mediate a variety of cellular processes by coupling transmembrane receptors to different effector molecules, including adenylyl cyclases and inositol-phospholipid-specific phospholipase C (PLC)1-3. Activation of adenylyl cyclases results in the production of cyclic AMP and activation of cAMP-dependent protein kinase (PKA). Phospholipase C catalyses the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP2) to generate diacylglycerol and inositol-1,4,5-triphosphate (InsP2), leading to the activation of protein kinase C (PKC) and the mobilization of intracellular calcium. The various PLC isoforms appear to be activated by different receptors, and in some cases by different G-protein components. There are four well-characterized forms of PLC-beta and all of them are activated to various extents by the G alpha q family of G proteins. Specific activation of PLC isoforms beta 2 and beta 3 by G-protein beta gamma subunits has also been reported. Although it has been suggested that PLC activity might be modulated by the adenylyl cyclase pathway, no clear link has been established between the two pathways. Here we report that cAMP-dependent protein kinase specifically inhibits G beta gamma-activated PLC-beta 2 activity but not that of the G alpha-activated PLC isoforms, and that the effect of PKA is not mimicked by PKC isozymes. Furthermore, we show that PKA directly phosphorylates serine residues of the PLC-beta 2 protein both in vivo and in vitro. Our results provide an insight into the specificity and nature of the crosstalk between the two G-protein-coupled signal transduction pathways.
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PMID:Regulation by cAMP-dependent protein kinease of a G-protein-mediated phospholipase C. 865 10

The action of angiotensin II (ANG II) was studied in single myocytes from rat portal vein, in which the cytoplasmic Ca++ concentration was estimated by emission from fluorescent dyes and the Ca++ channel current was measured with the whole-cell mode of the patch-clamp technique. ANG II stimulated Ca++ channel current through L-type Ca++ channels and initiated a slow and small increase in the cytoplasmic Ca++ concentration in cells in which intracellular Ca++ stores had been depleted by pretreatment with ryanodine and caffeine. Both Ca++ channel current stimulation and Ca++ responses were selectively inhibited by losartan, indicating activation of angiotensin AT1 receptors. Activation of Ca++ channels by ANG II was insensitive to treatment with pertussis toxin and cholera toxin. Intracellular applications of anti-G alpha q/alpha 11 and anti-phosphatidylinositol antibodies had no effect on the ANG II-induced stimulation of Ca++ channel current, indicating that phosphatidylinositol-specific phospholipase C was not involved in this signaling pathway. Down-regulation of protein kinase C and application of an inhibitor of protein kinase C blocked the ANG II-induced effects. Tricyclodecan-9-yl xanthogenate (an inhibitor of non-phosphatidylinositol-specific phospholipases C and phospholipases D) but not propranolol (an inhibitor of phospholipase D-derived diacylglycerol formation) suppressed the ANG II-induced effects. These data suggest that phosphatidylcholine-specific phospholipase C is involved in the ANG II signaling pathway leading to stimulation of L-type Ca++ channels by protein kinase C.
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PMID:Angiotensin II-mediated activation of L-type calcium channels involves phosphatidylinositol hydrolysis-independent activation of protein kinase C in rat portal vein myocytes. 876 93

The function of the phosphoinositide signal transduction system and the levels of heterotrimeric G-protein alpha-subunits were examined in postmortem prefrontal cortex regions (8/9) and region (10) from suicide victims with major depression and matched control subjects without psychiatric illness. The hydrolysis of [3H]phosphatidylinositol (PI) stimulated by phospholipase C, GTP-gamma-S, NaF, and neurotransmitter receptor agonists was measured in membrane preparations from both groups. Phospholipase C-beta activity was similar in depressed suicide and control subjects in the two regions of prefrontal cortex. In prefrontal cortex (10), but not in (8/9), the GTP-gamma-S concentration-dependent stimulation of [3H]PI hydrolysis was significantly lower (30%) in the depressed suicide group compared to the control group. Receptor-coupled, G-protein-mediated [3H]PI hydrolysis induced with carbachol, histamine, trans-1-aminocyclopentyl-1, 3-dicarboxylic acid (ACPD, a glutamatergic metabotropic receptor agonist), serotonin, or 2-methylthio-adenosine triphosphate (2mATP, a purinergic receptor agonist) in the presence of GTP-gamma-S stimulated equivalent responses in the two groups of subjects in each brain region. In prefrontal cortex (10) there was a 68% increase in the level of the 45 kDa subtype of G alpha s and in prefrontal cortex (8/9) there was a significant decrease (21%) in the level of G alpha i2 in the depressed suicide group compared to the control group. Levels of other heterotrimeric G-protein alpha-subunits (G alpha q/11, G alpha i1, and G alpha o) were not different in depressed suicide and control subjects in either brain region. Moreover, there were no differences in the levels of phospholipase C-beta or protein kinase C-alpha in the two groups of subjects in either brain region examined. These results demonstrate that in the prefrontal cortex of suicide victims with major depression compared to normal control subjects there is a region-specific alteration of G-protein-induced activation of the phosphoinositide signal transduction system and in the levels of G-protein alpha-subunits involved in cyclic AMP synthesis. These findings provide direct evidence in human brain that these two important signal transduction systems are altered in suicide subjects with major depression.
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PMID:Alterations in phosphoinositide signaling and G-protein levels in depressed suicide brain. 881 80


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