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The roles of multiple pathways in regulating bombesin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts.

ABSTRACT: The regulation of bombesin-stimulated phospholipase D (PLD) activity in Swiss 3T3 fibroblasts was examined. Increasing protein-tyrosine phosphorylation by using pervanadate to inhibit tyrosine phosphatases was found to stimulate protein kinase C (PKC)-independent [3H]phosphatidylbutanol ([3H]PtdBut) accumulation within 5 min, which continued to increase up to 30 min. The stimulation of PLD activity in response to submaximal [bombesin] could be decreased by approx. 50% by the tyrosine kinase inhibitor genistein, whereas pretreatment with genistein and the PKC inhibitor Ro-31-8220 completely abolished the generation of [3H]PtdBut in response to a maximal concentration of bombesin. The addition of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) into permeabilized cells resulted in an increase in [3H]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 microM GTP[S] did not increase the stimulation of PLD activity by any [bombesin] tested, whereas it was synergistic with that stimulated in response to phorbol 12-myristate 13-acetate. These findings suggest that bombesin-stimulated PLD activity is indirectly regulated by G-proteins, possibly through a kinase intermediate. Furthermore, activation of protein tyrosine kinases is proposed to account for the PKC-independent arm of bombesin-stimulated PLD activity. No evidence was obtained for a form of PLD directly regulated by tyrosine phosphorylation.

SUBMITTER: Briscoe CP

PROVIDER: S-EPMC1136489 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

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Rapid desensitization and resensitization of bombesin-stimulated phospholipase D activity in Swiss 3T3 cells.

Project description:The kinetics of bombesin-stimulated phospholipase D (PLD) activity were examined in Swiss 3T3 fibroblasts. The stimulated activity was found to rapidly desensitize, being completely absent after 40 s. This activity then quickly, but incompletely, resensitized, with PLD being detectable after a 4.5 min wash of the desensitized cells and 75-80% of the activity being recovered after 10 min. The desensitization was dose-dependent; however, the half-maximal stimulatory concentration of bombesin was an order of magnitude lower than that required for bombesin-stimulated second messenger generation and the KD for bombesin receptor binding. This suggested that desensitization was stimulated by a 'downstream' effect, but experiments have ruled out changes in protein kinase C activity and Ca2+ concentration. Binding experiments suggested that part of the desensitization is due to receptor internalization, and the requirement for an extracellular agonist for resensitization implies that receptor recycling plays a role. Over an extended time course, cycles of desensitization and resensitization of bombesin-stimulated PLD activity were apparent which may be relevant to mitogenic signalling. These studies add further evidence for a second messenger pathway of PLD activation, and the disparity between the kinetics of diacylglycerol generation and PLD activation supports the possibility that phosphatidic acid may have a messenger role in stimulated cells.

| S-EPMC1137983 | biostudies-other

Mass measurement of inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 mouse fibroblasts.

Project description:Two specific and selective assays were used to measure changes in the mass of Ins(1,4,5)P3 and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 cells. The results demonstrate that the increase in Ins(1,4,5)P3 was extremely rapid, but transient, returning to basal levels by 30 s. In contrast, the increase in sn-1,2-diacylglycerol was biphasic: the first phase mirrored the transient Ins(1,4,5)P3 response, whereas the second phase was sustained and occurred in the absence of elevated Ins(1,4,5)P3. The possible source of the second phase of diacylglycerol is discussed.

| S-EPMC1136930 | biostudies-other

Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein.

Project description:The stimulation of inositol phosphate generation by bombesin and GTP analogues was studied in Swiss 3T3 cells permeabilized by electroporation. Bombesin-stimulated inositol phosphate generation is potentiated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and inhibited by guanosine 5'-[beta-thio]diphosphate at all peptide concentrations tested, with no change in the EC50 value (concn. giving half-maximal response) for the agonist. Kinetic analysis showed that, although bombesin-stimulated [3H]InsP3 generation in [3H]inositol-labelled cells was rapid (maximal by 5-10 s), the response to GTP[S] alone displayed a distinct lag time of 20-30 s. This lag time was significantly decreased by the addition of bombesin, suggesting that in this system agonist-stimulated GTP/GDP exchange occurs. In addition, bombesin-stimulated generation of Ins(1,4,5)P3 mass at 10 s was enhanced by GTP[S] in the absence of a nucleotide response alone, a result consistent with this proposal. Pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent inhibition of bombesin-, but not GTP[S]-, stimulated inositol phosphate generation. Furthermore, although PMA pretreatment did not affect the lag time for InsP3 formation in response to GTP[S] alone, the degree of synergy between bombesin and the nucleotide was severely decreased at early time points. The results therefore demonstrate that the high-affinity bombesin receptor is coupled via a G-protein to phospholipase C in a manner consistent with a general model for receptor-G-protein interactions and that this coupling is sensitive to phosphorylation by protein kinase C.

| S-EPMC1131481 | biostudies-other

Phosphorylation and redistribution of the phosphatidylinositol-transfer protein in phorbol 12-myristate 13-acetate- and bombesin-stimulated Swiss mouse 3T3 fibroblasts.

Project description:By immunofluorescence microscopy it was shown that the phosphatidylinositol-transfer protein (PI-TP) becomes associated with the Golgi membranes when confluent (quiescent) Swiss mouse 3T3 fibroblast cells are stimulated with phorbol 12-myristate 13-acetate (PMA) and bombesin. Dibutyryl cyclic AMP or dexamethasone had no effect on the intracellular redistribution of PI-TP. In exponentially growing cells and in serum-starved (semi-quiescent) cells, PI-TP is already associated with Golgi structures. Stimulation of semi-quiescent cells by PMA resulted in a rapid redistribution of PI-TP. A similar yet slower response was observed after stimulation with bombesin. Stimulation of semi-quiescent 3T3 cells by PMA significantly increased the phosphorylation of PI-TP, as shown by immunoprecipitation of PI-TP from pre-labelled cells. No significant increase in phosphorylation of PI-TP was observed after stimulation of these cells by bombesin. Purified PI-TP was shown to be a substrate for protein kinase C in vitro. The possibility that the phosphorylation of PI-TP after activation of protein kinase C is involved in the observed redistribution of PI-TP is discussed.

| S-EPMC1132573 | biostudies-other

Multiple sources of sn-1,2-diacylglycerol in platelet-derived-growth-factor-stimulated Swiss 3T3 fibroblasts. Evidence for activation of phosphoinositidase C and phosphatidylcholine-specific phospholipase D.

Project description:Platelet-derived growth factor (PDGF) stimulated sn-1,2-diacylglycerol (DAG) mass formation in Swiss 3T3 fibroblasts with a lag time of some 30 s. The response was biphasic, with the second phase being sustained over time. PDGF also stimulated the formation of Ins(1,4,5)P3 with a similar lag time to the DAG response, suggesting that DAG is derived from PtdIns(4,5)P2 hydrolysis at this time point. PDGF-stimulated phosphatidylcholine (PtdCho) hydrolysis in Swiss 3T3 fibroblasts, as measured by the formation of water-soluble choline metabolites and phosphatidylbutanol (PtdBut) accumulation, was by a phospholipase D (PLD)-catalysed pathway which was kinetically downstream of initial PtdIns(4,5)P2 hydrolysis. Accumulation of PtdBut increased up to 15 min, suggesting that PLD activity is not rapidly densitized in response to PDGF. The kinetics of PtdCho hydrolysis closely paralleled the second phase of DAG formation, strongly suggesting that during prolonged stimulation periods PtdCho is a major source of DAG in these cells. However, since PtdIns(4,5)P2 breakdown was also prolonged, PDGF-stimulated DAG may be derived from both phospholipids. Down-regulation of protein kinase C (PKC), by pre-treatment with phorbol 12-myristate 13-acetate, abolished both [3H]choline and [3H]PtdBut formation, suggesting that PLD-catalysed PtdCho hydrolysis may be dependent on PKC activation, supporting its dependence on prior PtdIns(4,5)P2 hydrolysis.

| S-EPMC1151640 | biostudies-other

The regulation of phospholipase D activity and its role in sn-1,2-diradylglycerol formation in bombesin- and phorbol 12-myristate 13-acetate-stimulated Swiss 3T3 cells.

Project description:Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) to quiescent Swiss 3T3 cells resulted in a sustained increase in sn-1,2-diradylglycerol (DG) mass and [3H]DG in [3H]palmitate-labelled cells where phosphatidylcholine was the major labelled phospholipid. This occurred in the absence of inositol phosphate accumulation. In [3H]palmitate-labelled cells both bombesin and PMA stimulated the formation of phosphatidylbutanol ([3H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol. The kinetics of [3H]PtdBut formation were consistent with phospholipase D (PLD) activation preceding sustained DG formation. The inclusion of butan-1-ol inhibited 70% of PMA-stimulated DG formation but only 30% of the bombesin response. The ability of bombesin and PMA to stimulate the accumulation of [3H]PtdBut was completely abolished in Swiss 3T3 cells which had been pre-treated with 400 nM-PMA for 48 h to down-regulate protein kinase C activity. PMA-stimulated [3H]PtdBut formation was inhibited by 90% by the protein kinase C inhibitor Ro-31-8220 (10 microM), but bombesin-stimulated PtdBut accumulation was inhibited by at most 50% by the same concentration of inhibitor. Cyclic AMP-elevating agents, i.e. forskolin, dibutyryl cyclic AMP and isobutylmethylxanthine, did not inhibit bombesin stimulation of PLD activity. Bombesin-stimulated PLD activity was inhibited by 50% by buffering of the extracellular Ca2+ concentration to 150 nM, but combination of this treatment with Ro-31-8220 addition was less than additive. Ionophore A23187 alone was able to stimulate PLD activity, but this response was inhibited 50% by Ro-31-8220. Thapsigargin was unable to stimulate PLD activity and had no modulatory effect upon bombesin-stimulated PLD activity at any agonist concentration. The results are discussed in terms of the role of PLD in DG generation and the regulation of PLD activity both by bombesin and by PMA.

| S-EPMC1130566 | biostudies-other

Bombesin and epidermal growth factor stimulate the mitogen-activated protein kinase through different pathways in Swiss 3T3 cells.

Project description:Both bombesin and epidermal growth factor (EGF) are potent mitogens in Swiss 3T3 cells that nonetheless have dissimilar receptor structures. To explore possible common intracellular events involved in the stimulation of cellular growth by these two peptides, we have evaluated the regulation of the mitogen-activated protein (MAP) kinase. Exposure of Swiss 3T3 cells to bombesin, EGF or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) causes the rapid and transient stimulation of the enzyme activity. Pretreatment of cells with the protein kinase inhibitor H-7, or down-regulation of cellular protein kinase C by prolonged exposure to PMA, causes a decrease of over 90% in the activation of MAP kinase by bombesin. In contrast, these treatments have no effect on the stimulation of MAP kinase by EGF. The stimulation of MAP kinase activity by bombesin is dose-dependent, occurring over a narrow concentration range of the peptide. Both EGF and bombesin stimulate the phosphorylation of an immunoprecipitable MAP kinase protein migrating at 42 kDa on SDS/PAGE. Phosphoamino acid analysis of this phosphorylated protein reveals that EGF and bombesin stimulate phosphorylation on tyrosine, threonine and serine residues. Tyrosine phosphorylation of the enzyme, as evaluated by antiphosphotyrosine blotting of the immunoprecipitated protein, reveals that the time course of phosphorylation by both mitogens correlates with stimulation of enzyme activity. These results provide further evidence for the convergence of discrete pathways emanating from tyrosine kinase and G-protein-linked receptors in the regulation of MAP kinase.

| S-EPMC1132162 | biostudies-other

Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells.

Project description:Bombesin-related peptides stimulate a rapid increase in polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells. These peptides generate an increase in the efflux of 45Ca2+ from pre-labelled cells, a response consistent with an inositol trisphosphate-mediated mobilization of intracellular Ca2+. The bombesin-stimulated release of cellular 45Ca2+ is inhibited by tumour-promoting phorbol esters (e.g. 12-O-tetradecanoylphorbol 13-acetate, TPA). Although there are several possible sites of action at which this effect might occur, our results indicate that TPA induces an uncoupling of bombesin-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) without decreasing cellular binding of bombesin. In cultured cells, protein kinase C can be down-modulated by a prolonged incubation of the cells with phorbol esters. Such pretreatment greatly decreased the inhibitory effect of TPA on bombesin-stimulated PIP2 hydrolysis, suggesting that this action of the phorbol ester is mediated via protein kinase C. Since diacylglycerol is an endogenous activator of protein kinase C and a direct product of PIP2 hydrolysis, these results suggest that protein kinase C inhibition of polyphosphoinositide hydrolysis may function as a negative-feedback pathway. Cells in which protein kinase C has been down-modulated show elevated basal and bombesin-stimulated production of inositol phosphates, providing evidence that such a feedback loop limits polyphosphoinositide turnover in both unstimulated and mitogen-stimulated cells.

| S-EPMC1148179 | biostudies-other

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts.

Project description:The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

| S-EPMC2173402 | biostudies-literature

Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

Project description:Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant Gα(i) at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G(q) and G(i) families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα(11), a member of the G(q) family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα(13) was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G(s) was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα(i), but not Gα(q). We also show that PMT inhibits the GTPase activity of G(q).

| S-EPMC3489841 | biostudies-literature

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