scholarly journals Phorbol ester treatment of intact rabbit platelets greatly enhances both the basal and guanosine 5′-[γ-thio]triphosphate-stimulated phospholipase D activities of isolated platelet membranes. Physiological activation of phospholipase D may be secondary to activation of phospholipase C

1990 ◽  
Vol 271 (3) ◽  
pp. 693-700 ◽  
Author(s):  
J Van der Meulen ◽  
R J Haslam
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Kinase C ◽  
Gtp Binding ◽  
Platelet Membranes ◽  
Rabbit Platelets

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorbol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+free] of 1 microM, PLD activity was detected in control membranes, but was 5.4 +/- 0.8-fold (mean +/- S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 microM-guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated PLD by 18 +/- 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 +/- 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 microM-Ca2+ (3.5 +/- 0.2-fold and 1.8 +/- 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5′-[beta-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.

scholarly journals Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and a potentiation via activation of protein kinase C

1988 ◽  
Vol 253 (3) ◽  
pp. 711-719 ◽  
Author(s):  
I Magnaldo ◽  
J Pouysségur ◽  
S Paris
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Phospholipase C ◽  
Pertussis Toxin ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Kinase C ◽  
Gtp Binding

Previous studies in Chinese-hamster fibroblasts (CCL39 line) indicate that an important signalling pathway involved in thrombin's mitogenicity is the activation of a phosphoinositide-specific phospholipase C, mediated by a pertussis-toxin-sensitive GTP-binding protein (Gp). The present studies examine the effects of thrombin on the adenylate cyclase system and the interactions between the two signal transduction pathways. We report that thrombin exerts two opposite effects on cyclic AMP accumulation stimulated by cholera toxin, forskolin or prostaglandin E1. (1) Low thrombin concentrations (below 0.1 nM) decrease cyclic AMP formation. A similar inhibition is induced by A1F4-, and both thrombin- and A1F4- –induced inhibitions are abolished by pertussis toxin. (2) Increasing thrombin concentration from 0.1 to 10 nM results in a progressive suppression of adenylate cyclase inhibition and in a marked enhancement of cyclic AMP formation in pertussis-toxin-treated cells. A similar stimulation is induced by an active phorbol ester, and thrombin-induced potentiation of adenylate cyclase is suppressed by down-regulation of protein kinase C. Therefore, we conclude that (1) the inhibitory effect of thrombin on adenylate cyclase is the direct consequence of the activation of a pertussis-toxin-sensitive inhibitory GTP-binding protein (Gi) possibly identical with Gp, and (2) the potentiating effect of thrombin on cyclic AMP formation is due to stimulation of protein kinase C, as an indirect consequence of Gp activation. Our results suggest that the target of protein kinase C is an element of the adenylate cyclase-stimulatory GTP-binding protein (Gs) complex. At low thrombin concentrations, activation of phospholipase C is greatly attenuated by increased cyclic AMP, leading to predominance of the Gi-mediated inhibition.

Protein kinase C impairs the coupling of the GTP-binding protein to LTB4 receptor in neutrophil

1992 ◽  
Vol 44 (1) ◽  
pp. 11-24 ◽  
Author(s):  
P. Dryden ◽  
B. Allen ◽  
S. Wong ◽  
A. Reaney ◽  
T. Merali ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Kinase C ◽  
Gtp Binding ◽  
Ltb4 Receptor

Involvement of RhoA and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini

1999 ◽  
Vol 276 (4) ◽  
pp. G915-G923 ◽  
Author(s):  
Fumihiko Nozu ◽  
Yasuhiro Tsunoda ◽  
Adenike I. Ibitayo ◽  
Khalil N. Bitar ◽  
Chung Owyang
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Protein ◽  
Pancreatic Enzyme ◽  
Amylase Secretion ◽  
Gtp Binding Protein ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Gtp Binding ◽  
C3 Exoenzyme

We evaluated intracellular pathways responsible for the activation of the small GTP-binding protein Rho p21 in rat pancreatic acini. Intact acini were incubated with or without CCK and carbachol, and Triton X-100-soluble and crude microsomes were used for Western immunoblotting. When a RhoA-specific antibody was used, a single band at the location of 21 kDa was detected. CCK (10 pM–10 nM) and carbachol (0.1–100 μM) dose dependently increased the amount of immunodetectable RhoA with a peak increase occurring at 3 min. High-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE (1–1,000 nM) did not increase the intensities of the RhoA band, suggesting that stimulation of RhoA is mediated by the low-affinity CCK-A receptor. Although an increase in RhoA did not require the presence of extracellular Ca2+, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM abolished the appearance of the RhoA band in response to CCK and carbachol. The Gq protein inhibitor G protein antagonist-2A (10 μM) and the phospholipase C (PLC) inhibitor U-73122 (10 μM) markedly reduced RhoA bands in response to CCK. The protein kinase C (PKC) activator phorbol ester (10–1,000 nM) dose dependently increased the intensities of the RhoA band, which were inhibited by the PKC inhibitor K-252a (1 μM). The pp60c- src inhibitor herbimycin A (6 μM) inhibited the RhoA band in response to CCK, whereas the calmodulin inhibitor W-7 (100 μM) and the phosphoinositide 3-kinase inhibitor wortmannin (6 μM) had no effect. RhoA was immunoprecipitated with Src, suggesting association of RhoA with Src. Increases in mass of this complex were observed with CCK stimulation. In permeabilized acini, the Rho inhibitor Clostridium botulinum C3 exoenzyme dose dependently inhibited amylase secretion evoked by a Ca2+ concentration with an IC50 of C3 exoenzyme at 1 ng/ml. We concluded that the small GTP-binding protein RhoA p21 exists in pancreatic acini and appears to be involved in the mediation of pancreatic enzyme secretion evoked by CCK and carbachol. RhoA pathways are involved in the activation of PKC and Src cascades via Gq protein and PLC.

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