Protein kinase C-promoted inhibition of Galpha(11)-stimulated phospholipase C-beta activity.
The effects of protein kinase C (PKC) activation on inositol lipid signaling were examined. Using the turkey erythrocyte model of receptor-regulated phosphoinositide hydrolysis, we developed a membrane reconstitution assay to study directly the effects of activation of PKC on the activities of Galpha(11), independent of potential effects on the receptor or on PLC-beta. Membranes isolated from erythrocytes pretreated with 4beta-phorbol-12beta-myristate-13alpha-acetate (PMA) exhibited a decreased capacity for Galpha(11)-mediated activation of purified, reconstituted PLC-beta1. This inhibitory effect was dependent on both the time and concentration of PMA incubation and occurred as a decrease in the efficacy of GTPgammaS for activation of PLC-beta1, both in the presence and absence of agonist; no change in the apparent affinity for the guanine nucleotide occurred. Similar inhibitory effects were observed after treatment with the PKC activator phorbol-12,13-dibutyrate but not after treatment with an inactive phorbol ester. The inhibitory effects of PMA were prevented by coaddition of the PKC inhibitor bisindolylmaleimide. Although the effects of PKC could be localized to the membrane, no phosphorylation of Galpha(11) occurred either in vitro in the presence of purified PKC or in intact erythrocytes after PMA treatment. These results support the hypothesis that a signaling protein other than Galpha(11) is the target for PKC and that PKC-promoted phosphorylation of this protein results in a phosphorylation-dependent suppression of Galpha(11)-mediated PLC-beta1 activation.
University of North Carolina School of Medicine, Department of Pharmacology, Chapel Hill, North Carolina, USA. cunnml@med.unc.edu
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Cunningham
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Filtz
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Harden
D000818:Animals; D004910:Erythrocyte Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054799:Phospholipase C beta; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014422:Turkeys; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D007527:Isoenzymes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011493:Protein Kinase C; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate