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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chemoattractant cAMP induces directed cell locomotion in Dictyostelium cells. Several second messenger pathways are activated upon binding of cAMP to G-protein-coupled receptors, including adenylyl cyclase, guanylyl cyclase, phospholipase C, and the opening of plasma membrane Ca2+ channels. These second messenger responses are unaltered in many chemotactic mutants, except for the cGMP response. Activation of guanylyl cyclase depends on G-proteins and is regulated by a cGMP-binding protein in a complex manner. This cGMP-binding protein also mediates intracellular functions of cGMP to activate a PKC-related kinase that phosphorylates myosin II heavy chain, thereby allowing myosin filaments to rearrange during cell movement.
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PMID:cGMP as second messenger during Dictyostelium chemotaxis. 924 16

During the developmental life cycle of the cellular slime mould Dictyostelium discoideum cells aggregate in response to pulses of extracellular cAMP. This chemotactic agent stimulates a number of signalling pathways in the cell including the activation of a phospholipase C activity leading to the transient generation of inositol 3,4,5-trisphosphate and diacylglycerol. The role of diacylglycerol in chemotactic response and development of Dictyostelium is not known. We have evidence to suggest that two protein kinase C-like enzymes exist in Dictyostelium due to the different cellular responses to two inhibitors specific for protein kinase C. One enzyme is preferentially sensitive to D-erythro-sphingosine, a diacylglycerol analogue, and is required for growth. A second is preferentially inhibited by bisindolylmaleimide GF109203X and is required for chemotaxis. We have identified protein kinase C-like kinase activity in Dictyostelium cell extracts which appears as the cells aggregate. This activity is stimulated by diacylglycerol, especially biologically relevant diacylglycerol species, and phosphorylates a peptide substrate which is an efficient substrate for mammalian protein kinase Cs. This activity is a candidate for the effector of diacylglycerol generated during the aggregative phase of Dictyostelium development and defines a role for diacylglycerol in the chemotactic response.
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PMID:A protein kinase C-like activity involved in the chemotactic response of Dictyostelium discoideum. 942 Nov 98

When the unicellular eukaryote Dictyostelium discoideum starves, it senses the local density of other starving cells by simultaneously secreting and sensing a glycoprotein called conditioned medium factor (CMF). When the density of starving cells is high, the corresponding high density of CMF permits signal transduction through cAR1, the chemoattractant cAMP receptor. cAR1 activates a heterotrimeric G protein whose alpha-subunit is Galpha2. CMF regulates cAMP signal transduction in part by regulating the lifetime of the cAMP-stimulated Galpha2-GTP configuration. We find here that guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) inhibits the binding of CMF to membranes, suggesting that the putative CMF receptor is coupled to a G protein. Cells lacking Galpha1 (Galpha1 null) do not exhibit GTPgammaS inhibition of CMF binding and do not exhibit CMF regulation of cAMP signal transduction, suggesting that the putative CMF receptor interacts with Galpha1. Work by others has suggested that Galpha1 inhibits phospholipase C (PLC), yet when cells lacking either Galpha1 or PLC were starved at high cell densities (and thus in the presence of CMF), they developed normally and had normal cAMP signal transduction. We find that CMF activates PLC. Galpha1 null cells starved in the absence or presence of CMF behave in a manner similar to control cells starved in the presence of CMF in that they extend pseudopods, have an activated PLC, have a low cAMP-stimulated GTPase, permit cAMP signal transduction, and aggregate. Cells lacking Gbeta have a low PLC activity that cannot be stimulated by CMF. Cells lacking PLC exhibit IP3 levels and cAMP-stimulated GTP hydrolysis rates intermediate to what is observed in wild-type cells starved in the absence or in the presence of an optimal amount of CMF. We hypothesize that CMF binds to its receptor, releasing Gbetagamma from Galpha1. This activates PLC, which causes the Galpha2 GTPase to be inhibited, prolonging the lifetime of the cAMP-activated Galpha2-GTP configuration. This, in turn, allows cAR1-mediated cAMP signal transduction to take place.
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PMID:Cell density sensing mediated by a G protein-coupled receptor activating phospholipase C. 952 20

cAMP-induced Ca2+ fluxes in Dictyostelium discoideum largely depend on phospholipase A2 activity generating non-esterified fatty acids [Schaloske and Malchow (1997) Biochem. J. 327, 233-238]. In the present study the effect of fatty acids on Ca2+ homoeostasis in D. discoideum was investigated. Cytosolic free Ca2+ concentration ([Ca2+]i) was analysed by digital imaging of single fura2-dextran-loaded cells. Arachidonic acid and linoleic acid induced a transient increase in [Ca2+]i. The concentration of arachidonic acid determined the percentage of responding cells, with the mean height of the increase being dose-independent. In nominally Ca2+-free medium or in the presence of bis-(o-aminophenoxy)ethane-N, N,N',N'-tetra-acetic acid (BAPTA), no [Ca2+]i transient was detectable. In spite of this, we found that (1) arachidonic acid induced Ca2+ release from permeabilized cells and from vesicular fractions at concentrations that elicited Ca2+ influx in intact cells and (2) Ca2+ entry was inhibited by inhibitors of Ca2+-transport ATPases and V-type H+-ATPase, indicating that intracellular Ca2+ release precedes Ca2+ entry. Inhibition studies and mutant analysis point to the acidosomal Ca2+ stores as a target of fatty acids. Although fatty acids can substitute fully for cAMP with respect to Ca2+ influx in wild-type cells, experiments with a mutant strain revealed that cAMP also sensitizes the Ca2+-entry mechanism: cAMP-induced Ca2+ influx was normal in a phospholipase C knockout mutant but influx was fairly insensitive to arachidonic acid in this strain. This defect could be overcome by higher doses of arachidonic acid which cause sufficient Ca2+ to be released from the stores to trigger extracellular Ca2+ entry.
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PMID:Fatty acids induce release of Ca2+ from acidosomal stores and activate capacitative Ca2+ entry in Dictyostelium discoideum. 960 Oct 85

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein beta subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type beta subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In beta subunit-null cells, and in a series of beta subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein-actin transfected cells showed that beta subunit- null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.
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PMID:G protein beta subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton. 964 46

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C-specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.
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PMID:The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum. 995 Jun 84

The heterotrimeric G protein, G2, from the eukaryotic organism Dictyostelium discoideum participates in signal transduction pathways which are essential to Dictyostelium's developmental life cycle. G2 is activated by cell surface cAMP receptors and in turn is required for the activation of a host of effectors, including adenylyl cyclase, guanylyl cyclase, and phospholipase C. Myristoylation of G protein alpha-subunits is known to affect alpha-subunit association with the beta gamma subunits and membrane localization. The putative site for N-terminal myristoylation of G alpha 2 was mutated from Gly to Ala (G2A) and expressed in the g alpha 2-null cell line, MYC2. Transformants expressing G alpha 2-G2A exhibit physiological and biochemical changes from wild-type cells. G alpha 2-G2A expressing cells fail to rescue the aggregation-minus phenotype of MYC2 cells on developmental agar plates. G alpha 2-G2A expressing cells are also not chemotactic to cAMP in a standard drop assay. G alpha 2-WT is found in both the pellet and supernatant fractions following lysis of the cells. G alpha 2-G2A however is found almost exclusively in the lysate supernatant. G alpha 2 is radiolabeled upon incubation of cells in [3H]myristate, while G alpha 2-G2A is not labeled. Examination of activation of the effectors adenylyl cyclase and guanylyl cyclase reveals that G alpha 2-G2A expressing cells partially activate adenylyl cyclase but show no cAMP-stimulation of guanylyl cyclase. The physiological deviations from wild-type can be explained by the variations in effector activation, possibly due to improper localization of the non-myristoylated G alpha 2-G2A to the cytosol.
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PMID:Aggregation of Dictyostelium discoideum is dependent on myristoylation and membrane localization of the G protein alpha-subunit, G alpha 2. 1040 98

Soluble phosphatidylinositol transfer proteins (PITPs) have important roles in lipid-mediated signalling as well as in membrane traffic. Two PITPs (alpha and beta) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene). However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells. We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene. This protein, DdPITP1, is homologous with mammalian PITPalpha and PITPbeta. We have also cloned a second gene (DdPITP2) related in sequence to DdPITP1. In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs. DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PtdIns from one membrane compartment to another; they thus exhibited the hallmark of PITPs. Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis. The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of protein with genetic approaches.
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PMID:Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae sec14p are found in the same cell. 1076 90

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.
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PMID:The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence. 1128 75

A cell adhesion molecule, 80-kDa csA, is involved in EDTA-resistant cell contact at the aggregation stage of Dictyostelium discoideum. A 31-kDa csA was isolated from the 80-kDa csA by treatment with Achromobacter protease I. Results from thin-layer chromatography and MALDI-TOF MS analysis indicated that the 31-kDa csA contains ceramide as a component of glycosylphosphatidyl-inositol (GPI). Comparison between the 80-kDa csA and the 31-kDa csA treated with phosphatidylinositol-specific phospholipase C (PI-PLC) or GPI-specific phospholipase D (GPI-PLD) was carried out. Our results indicated that the GPI-anchor of the 31-kDa csA was more sensitive to PI-PLC treatment than that of the 80-kDa csA, and that the anchor in both was easily cleaved by GPI-PLD treatment. They suggested that the resistance of 80-kDa csA to PI-PLC treatment was due to steric hindrance and myo-inositol modification. The results of the 80-kDa csA and the 31-kDa csA treated with sphingomyelinase were similar to those with PI-PLC treatment. In the presence of 1,10-phenanthroline, a GPI-PLD inhibitor, development of Dictyostelium was markedly inhibited, suggesting that GPI-PLD is functional in developmental regulation through cell adhesion.
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PMID:Cleavage with phospholipase of the lipid anchor in the cell adhesion molecule, csA, from Dictyostelium discoideum. 1638 74


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