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: UNIPROT:P15088 (mast cell)
14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We recently demonstrated that the activation of ceramide kinase (CERK) and the formation of its product, ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca(2+) (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca(2+) as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca(2+)-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca(2+). The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM.CERK complexes. An in vitro CaM binding assay also demonstrated Ca(2+)-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca(2+) sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca(2+)-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca(2+)-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein kinase II, which had been assumed to be the main target of CaM in mast cells.
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PMID:Calmodulin is involved in the Ca2+-dependent activation of ceramide kinase as a calcium sensor. 1620 36

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells results in the production and secretion of sphingosine 1-phosphate (S1P), a lipid metabolite present in the lungs of allergen-challenged asthmatics. Herein we report that two isoforms of sphingosine kinase (SphK1 and SphK2) are expressed and activated upon FcepsilonRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcepsilonRI coupling to SphK1 and -2 and for subsequent S1P production. Normal activation of SphK1 and -2 was restored by expression of wild type Fyn but only partly with a kinase-defective Fyn, indicating that induction of SphK1 and SphK2 depended on both catalytic and noncatalytic properties of Fyn. Downstream of Fyn, the requirements for SphK1 activation differed from that of SphK2. Whereas SphK1 was considerably dependent on the adapter Grb2-associated binder 2 and phosphatidylinositol 3-OH kinase, SphK2 showed minimal dependence on these molecules. Fyn-deficient BMMC were defective in chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S1P to FcepsilonRI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.
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PMID:IgE-dependent activation of sphingosine kinases 1 and 2 and secretion of sphingosine 1-phosphate requires Fyn kinase and contributes to mast cell responses. 1631 95

Antigen-induced degranulation of mast cells plays a pivotal role in allergic and inflammatory responses. Recently, ceramide kinase (CERK) and its phosphorylated product ceramide 1-phosphate (C1P) have emerged as important players in mast cell degranulation. Here, we describe the synthesis of a novel F-12509A olefin isomer, K1, as an effective CERK inhibitor. In vitro kinase assays demonstrated that K1 effectively inhibits CERK without inhibiting sphingosine kinase and diacylglycerol kinase. Treating RBL-2H3 cells with K1 reduced cellular C1P levels to 40% yet had no effect on cell growth. Furthermore, treatment with K1 significantly suppressed both calcium ionophore- and IgE/antigen-induced degranulation, indicating that K1 interferes with signals that happen downstream of Ca(2+) mobilization. Finally, we show that K1 affects neither IgE/antigen-induced global tyrosine phosphorylation nor subsequent Ca(2+) elevation, suggesting a specificity for CERK-mediated signals. Our novel CERK inhibitor provides a useful tool for studying the biological functions of CERK and C1P. Moreover, to our knowledge, this is the first report demonstrating that inhibition of CERK suppresses IgE/antigen-induced mast cell degranulation. This finding suggests that CERK inhibitors might be a potential therapeutic tool in the treatment of allergic diseases.
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PMID:Suppression of mast cell degranulation by a novel ceramide kinase inhibitor, the F-12509A olefin isomer K1. 1635 67

The membrane phospholipid phosphatidylinositol 4, 5-bisphosphate [PI (4,5)P2] is a critical signal transducer in eukaryotic cells. However, the physiological roles of the type I phosphatidylinositol phosphate kinases(PIPKIs) that synthesize PI(4,5) P2 are unknown. Here we show that the alpha isozyme of PIPKI (PIPKIalpha) negatively regulates mast cell functions and anaphylactic responses. In vitro, PIPKIalpha-deficient mast cells exhibit increased degranulation and cytokine production after Fc(epsilon)RI crosslinking. In vivo, PIPKIalpha-/- mice display enhanced passive cutaneous and systemic anaphylaxis. Mechanistically, filamentous actin was diminished in PIPKIalpha-/- mast cells and enhanced degranulation observed in the absence of PIPKIa was phenocopied in wild type mast cells treated with latrunculin, a pharmacological inhibitor of actin polymerization. Moreover, the association of Fc(epsilon)RI with lipid rafts and Fc(epsilon)RI mediated activation of signaling proteins is augmented in PIPKIalpha-/- mast cells. PIPKIalpha is thus a negative regulator of Fc(epsilon)RI-mediated cellular responses and anaphylaxis which functions by controlling the actin cytoskeleton and dynamics of Fc(epsilon)RI signaling. Our results are thus the first genetic evidence that PIPKI isoforms, and the pool of PI (4,5) P2 produced by each isoform, may be functionally specialized.
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PMID:[Compartmentalization of phosphatidylinositol 4, 5-bisphosphate]. 1649 29

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including sphingosine-1-phosphate (S1P). However, it is not known how S1P is released from cells. Here, we report that S1P is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated S1P release was inhibited by MK571, an inhibitor of ABCC1 (MRP1), but not by inhibitors of ABCB1 (MDR-1, P-glycoprotein). Moreover, down-regulation of ABCC1 with small interfering RNA, which decreased its cell surface expression, markedly reduced S1P export from both rat RBL-2H3 and human LAD2 mast cells. Transport of S1P by ABCC1 influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S1P functions in mast cell-mediated immune responses.
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PMID:Role of ABCC1 in export of sphingosine-1-phosphate from mast cells. 1705 Jun 92

Sphingosine-1-phosphate, a key mediator in immune cell trafficking, is elevated in the lungs of asthmatic patients and regulates pulmonary epithelium permeability. Stimulation of mast cells by allergens induces two mammalian sphingosine kinases (Sphk1 and Sphk2) to produce sphingosine-1-phosphate (S1P). Little is known about the individual role of these kinases in regulating immune cell function. Here we show that in mast cells, Sphk2 is required for production of S1P, for calcium influx, for activation of protein kinase C, and for cytokine production and degranulation. However, susceptibility to in vivo anaphylaxis is determined both by S1P within the mast cell compartment and by circulating S1P generated by Sphk1 predominantly from a non-mast cell source(s). Thus, sphingosine kinases are determinants of mast cell responsiveness, demonstrating a previously unrecognized relationship with anaphylaxis.
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PMID:The sphingosine kinase-sphingosine-1-phosphate axis is a determinant of mast cell function and anaphylaxis. 1737 88

Generation of sphingosine-1-phosphate by sphingosine kinases (SphKs) promotes allergic inflammatory diseases, but the roles of the individual SphK isoforms are unclear. Olivera et al. (2007) show that SphK2 regulates mast cell activation whereas SphK1 enhances susceptibility to antigen challenge in vivo.
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PMID:Division of labor: specialization of sphingosine kinases in mast cells. 1734 96

The Src family kinases Fyn and Lyn are important modulators of the molecular events initiated by engagement of the high-affinity IgE receptor (Fc epsilon RI). These kinases control many of the early signaling events and initiate the production of several lipid metabolites that have an important role in regulating mast cell responses. The intracellular level of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), which is produced by phosphatidylinositol 3-OH kinase, plays an important role in determining the extent of a mast cells response to a stimulus. Enhanced levels lead to a hyperdegranulating phenotype (as seen in SHIP-1(-/-) and Lyn(-/-) mast cells), whereas decreased levels cause hypodegranulation (as seen in Fyn(-/-) mast cells). Downregulation of mast cell phosphatase and tensin homologue deleted on chromosone 10 expression, a phosphatase that reduces cellular levels of PIP(3), caused constitutive cytokine production, demonstrating that this response is particularly sensitive to PIP(3) levels. Lyn and Fyn are also intimately linked to other lipid kinases, like sphingosine kinases (SphK). By producing sphingosine-1-phosphate (S1P), SphKs contribute to mast cell chemotaxis and degranulation. In vivo studies now reveal that circulating S1P as well as that found within the mast cell is important in determining mast cell responsiveness. These studies demonstrate the connection between Src protein tyrosine kinases and lipid second messengers that control mast cell function and allergic responses.
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PMID:Src family kinases and lipid mediators in control of allergic inflammation. 1749 64

Ceramide kinase and its product ceramide 1-phosphate have been implicated in cellular proliferation and survival, activation of cytosolic phospholipase A(2), mast cell degranulation, and phagocytosis. Current assays for ceramide kinase activity employ [(32)P]ATP, with separation of labeled product from excess ATP by organic extraction and thin-layer chromatography. We have developed a fluorescent plate reader assay for ceramide kinase that uses commercially available C6-NBD ceramide (N-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-D-erythro-sphingosine). Our assay is based on the differential partitioning of substrate and product following a single chloroform/methanol extraction. The product, which partitions into the aqueous phase at physiological pH, is quantitated directly in a plate reader. The substrate may be delivered using either fatty acid-free albumin or detergent/lipid mixed micelles, and we have found that the use of albumin rather than detergent micelles allows one to detect lipid interactions with the enzyme that might otherwise go unnoticed. Our method is useful for assaying ceramide kinase activity both in vitro and in cultured cells, and it offers several advantages over the conventional assay, including greater speed, the ability to run a larger number of assay replicates at one time, and the elimination of environmental and safety issues associated with the use of radioactive materials.
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PMID:A fluorescent plate reader assay for ceramide kinase. 1820 78

Nearly 25 years ago the first function of an inositol phosphate, namely Ins(1,4,5)P3, was reported to act as a "second messenger" to mobilize calcium from the endoplasmic reticulum (ER). Since this discovery, many other inositol phosphates and the kinases and phosphatases that generate these inositol phosphates have subsequently been discovered. However, the function of these "higher order" inositol phosphates in biological processes, if any, has remained a mystery. Interest in higher order inositol phosphates, such as Ins(1,3,4,5)P4, was renewed this year following reports of novel roles for these molecules in distinct processes within the immune system ranging from T cell development, B cell development and tolerance induction, as well as neutrophil and mast cell function. In this review, we will touch upon recent advances in inositol phosphate function in mammalian cells. More specifically, we will highlight new studies that have identified novel functions for specific higher order inositol phosphates, such as Ins(1,3,4,5)P4, in the immune system.
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PMID:Beyond IP3: roles for higher order inositol phosphates in immune cell signaling. 1823 37


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