Synergistic activation of phospholipase D by protein kinase C- and G-protein-mediated pathways in streptolysin O-permeabilized HL60 cells.
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ABSTRACT:
SUBMITTER: Geny B
PROVIDER: S-EPMC1132670 | biostudies-other | 1992 Jun
REPOSITORIES: biostudies-other
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Project description:The possible role of tyrosine phosphorylation in the activation of granulocytic HL60 cells was examined using vanadate, a phosphotyrosine phosphatase inhibitor. Treatment of permeabilized cells with micromolar concentrations of vanadate resulted in a substantial accumulation of tyrosine-phosphorylated proteins, detected by immunoblotting. At comparable concentrations, vanadate was also found to elicit an NADPH-dependent burst of oxygen utilization. Actin assembly, studied using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, was similarly stimulated by vanadate, though considerably higher concentrations were required to observe this effect. In contrast with these responses, the secretion of lysozyme was not stimulated by vanadate, nor did vanadate affect calcium-induced secretion. Therefore, accumulation of tyrosine-phosphorylated proteins is associated with stimulation of some, but not all, of the responses characteristic of granulocytic cell activation. This indicates that the effects of vanadate are selective and suggests divergence of the signalling pathways leading to the individual effectors.
| S-EPMC1131541 | biostudies-other
Project description:The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5'-[beta gamma-imido]-triphosphate greater than guanosine 5'-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.
| S-EPMC1148866 | biostudies-other
Project description:BackgroundProtein kinase D (PKD) constitutes a novel family of serine/threonine protein kinases implicated in fundamental biological activities including cell proliferation, survival, migration, and immune responses. Activation of PKD in these cellular activities has been linked to many extracellular signals acting through antigen receptor engagement, receptor tyrosine kinases, as well as G protein-coupled receptors. In the latter case, it is generally believed that the G? subunits of the Gq family are highly effective in mediating PKD activation, whereas little is known with regard to the ability of G?? dimers and other G? subunits to stimulate PKD. It has been suggested that the interaction between G?? and the PH domain of PKD, or the G??-induced PLC?/PKC activity is critical for the induction of PKD activation. However, the relative contribution of these two apparently independent events to G??-mediated PKD activation has yet to be addressed.ResultsIn this report, we demonstrate that among various members in the four G protein families, only the G? subunits of the Gq family effectively activate all the three PKD isoforms (PKD1/2/3), while G? subunits of other G protein families (Gs, Gi, and G12) are ineffective. Though the G? subunits of Gi family are unable to stimulate PKD, receptors linked to Gi proteins are capable of triggering PKD activation in cell lines endogenously expressing (HeLa cells and Jurkat T-cells) or exogenously transfected with (HEK293 cells) G??-sensitive PLC?2/3 isoforms. This indicates that the Gi-mediated PKD activation is dependent on the released G?? dimers upon stimulation. Further investigation on individual G?? combinations (i.e. G?1 with G?1-13) revealed that, even if they can stimulate the PLC? activity in a comparable manner, only those G?1? dimers with ?2, ?3, ?4, ?5, ?7, and ?10 can serve as effective activators of PKD. We also demonstrated that Gi-mediated PKD activation is essential for the SDF-1?-induced chemotaxis on Jurkat T-cells.ConclusionsOur current report illustrates that G?? dimers from the Gi proteins may activate PKD in a PLC?2/3-dependent manner, and the specific identities of G? components within G?? dimers may determine this stimulatory action.
| S-EPMC3637504 | biostudies-literature
Project description:Selectins mediate leukocyte rolling, trigger beta(2)-integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) gamma2- and phosphoinositide 3-kinase (PI3K) gamma-dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP(3)), and inducing E-selectin-mediated slow rolling. Inhibition of this signal-transduction pathway diminished Galpha(i)-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Galpha(i)-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk(-/-) and Plcg2(-/-) mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement.
| S-EPMC2858472 | biostudies-literature
Project description:Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA). Ca2+ alone in the range 0.5-1.0 microM also induced gamma-chain phosphorylation in some T-lymphoblast preparations; that in Jurkat-6 cells occurred at lower concentrations (50-500 nM). Two experimental approaches were used to investigate the possible involvement of PKC. Firstly, when permeabilization was carried out in buffer lacking free Ca2+, PKC was lost from the cells, and gamma-chain phosphorylation could then no longer be induced on subsequent addition of Pdbu or PHA in 400 nM-Ca2+, or 800 nM-Ca2+ alone, to permeabilized cells. However, when permeabilization was carried out in the presence of these three agents, PKC was translocated to intracellular membranes, and subsequent addition of [gamma-32P]ATP to these cells then resulted in gamma-chain phosphorylation. In the second approach, induction of gamma-chain phosphorylation by Pdbu, 1-oleoyl-2-acetylglycerol, 1,2-diolein, PHA or Ca2+ alone was effectively blocked by permeabilizing T cells in the presence of a PKC pseudosubstrate peptide (50 microM). Pseudosubstrate concentrations in the range 7-20 microM inhibited gamma-chain phosphorylation by 50%. In contrast, addition of four other 'irrelevant' basic peptides (50 microM) did not result in detectable inhibition, and 50 microM-pseudosubstrate did not inhibit the phosphorylation of 17 other polypeptides isolated from permeabilized T cells. These data suggest that Pdbu-, 1,2-diacylglycerol-, PHA- and Ca2+-induced phosphorylation of the CD3-antigen gamma chain in permeabilized T cells is mediated by PKC.
| S-EPMC1138760 | biostudies-other
Project description:Receptor-directed agonists including N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), C5a, ATP and UTP all activate phospholipase D (PLD), which is accompanied by secretion in differentiated HL60 cells. Interference in the production of phosphatidase (PA) by the PLD pathway by diverting it towards the production of phosphatidylethanol (PEt) in the presence of ethanol leads to near-total inhibition of the secretion evoked by ATP and UTP and a partial inhibition of that evoked by fMetLeuPhe and C5a. In streptolysin-O-permeabilized cells, fMetLeuPhe is able to activate PLD, and this is dependent on the presence of a low concentration of guanosine 5'-[gamma-thio]-triphosphate (GTP[S]). Ca2+ (10 microM) and GTP[S] individually or in combination are also able to activate PLD and secretion. The stimulation of secretion in permeabilized cells stimulated by Ca2+ alone or fMetLeuPhe or GTP[S] is also abrogated when the production of PA is diverted to PEt by the presence of ethanol. Activation of PLD by GTP[S] or fMetLeuPhe is decreased if the cells are permeabilized first and GTP[S] or fMetLeuPhe is added subsequently. This corresponds well with the loss of the secretory response. We conclude that the ability of GTP[S] or fMetLeuPhe to stimulate secretion from permeabilized cells is dependent on a prior activation of the PLD signalling pathway. PA, generated as a consequence of PLD activation, acts as second messenger that can provide an initiating signal for secretion and is not required for exocytosis itself.
| S-EPMC1134415 | biostudies-other
Project description:It has been established that protein kinase Czeta (PKCzeta) participates in diverse signaling pathways and cellular functions in a wide variety of cells, exhibiting properties relevant to cellular survival and proliferation. Currently, however, the regulation mechanism of PKCzeta remains elusive. Here, for the first time, we determine that phospholipase D2 (PLD2) enhances PKCzeta activity through direct interaction in a lipase activity-independent manner. This interaction of the PLD2-Phox homology (PX) domain with the PKCzeta-kinase domain also induces the activation loop phosphorylation of PKCzeta and downstream signal stimulation, as measured by p70 S6 kinase phosphorylation. Furthermore, only the PLD2-PX domain directly stimulates PKCzeta activity in vitro, and it is necessary for the formation of the ternary complex with phosphoinositide-dependent kinase 1 and PKCzeta. The mutant that substitutes the triple lysine residues (Lys101, Lys102, and Lys103) within the PLD2-PX domain with alanine abolishes interaction with the PKCzeta-kinase domain and activation of PKCzeta. Moreover, breast cancer cell viability is significantly affected by PLD2 silencing. Taken together, these results suggest that the PLD2-mediated PKCzeta activation is induced by its PX domain performing both direct activation of PKCzeta and assistance of activation loop phosphorylation. Furthermore, we find it is an important factor in the survival of breast cancer cells.
| S-EPMC1069590 | biostudies-literature
Project description:Activation of protein kinase C (PKC) in human T lymphocytes is an immediate consequence of mitogenic signalling via the antigen-receptor complex and CD2 antigen. In order to investigate further the signal-transduction pathways which result in PKC activation, we have established a novel PKC assay system using streptolysin-O-permeabilized T cells. Known peptide substrates of PKC were introduced into permeabilized cells in the presence of [gamma-32P]ATP, 3 mM-Mg2+ and 150 nM free Ca2+. The peptide found to have the lowest background phosphorylation had the sequence Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys (peptide GS), and the phosphorylation of the peptide was increased up to 6-fold by direct activation of PKC with phorbol 12,13-dibutyrate. Induction of PKC activation with the UCHT1 antibody against the CD3 antigen, or with phytohaemagglutinin (PHA) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), increased peptide-GS phosphorylation by 2-3 fold. The specificity of PKC action on peptide GS was demonstrated by blocking increases in phosphorylation with a pseudosubstrate peptide PKC inhibitor. PKC activation by this technique could be detected within 1 min of adding external ligand. Dose-response curves revealed that PHA-induced production of inositol phosphates correlated closely with PKC activities, whereas only a partial correlation between these parameters was observed with GTP[S]. Our data are consistent with the presence of more than one G-protein-mediated pathway of PKC regulation in T cells. The quantitative PKC assay system described is both simple and reproducible, and its potential application to a wide range of cell types should prove useful in further investigations of PKC activation mechanisms.
| S-EPMC1131432 | biostudies-other
Project description:In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.
| S-EPMC2590688 | biostudies-literature
Project description:Human platelets containing dense granules labelled with 5-hydroxy[14C]tryptamine ([14C]5-HT) were permeabilized by exposure to streptolysin O (SLO) in the presence of 4 mM [gamma-32P]ATP. Addition of either 100 nM phorbol 12-myristate 13-acetate (PMA) or of Ca2+ (pCa 5) at the same time as SLO induced secretion of dense-granule [14C]5-HT and the phosphorylation of pleckstrin by protein kinase C (PKC). Ca2+ also induced phosphorylation of myosin P-light chains. Guanosine 5'-[gamma-thio]triphosphate (GTP[S], 100 microM) did not stimulate secretion from SLO-permeabilized platelets in the absence of Ca2+ (pCa>9), but greatly potentiated secretion in the presence of low PMA (10 nM) or low Ca2+ (pCa 6). However, GTP[S] did stimulate myosin P-light-chain phosphorylation in the absence of Ca2+, an effect that was associated with morphological changes, including granule centralization. Inhibition of PKC and of pleckstrin phosphorylation by Ro 31-8220 blocked secretion induced by PMA or by GTP[S] and PMA in the absence of Ca2+, but did not prevent the GTP[S]-induced phosphorylation of myosin P-light chains or secretion induced by Ca2+ at pCa 5. When the time period between exposure of platelets to SLO and challenge at pCa>9 with PMA or with GTP[S] and PMA was increased, there were rapid and parallel decreases in the secretion and pleckstrin phosphorylation responses, which were lost after 3-5 min. In contrast, the responsiveness of secretion to Ca2+ (pCa 5) or to GTP[S] and Ca2+ (pCa 6) persisted for at least 10 min after exposure of platelets to SLO, although the ability of pleckstrin to undergo phosphorylation was still lost after 3-5 min. Both PKC and pleckstrin were undetectable within platelets after 5 min exposure to SLO. The results suggest that the loss of responsiveness to PMA or to GTP[S] and PMA is attributable to the leakage of PKC (and possibly pleckstrin) from the platelets, whereas secretion stimulated by Ca2+ or by GTP[S] and Ca2+ utilizes membrane-associated Ca2+- and GTP-binding proteins and occurs independently of PKC activation.
| S-EPMC1218881 | biostudies-other