Abstract
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 Gα11, independent of potential effects on the receptor or on PLC-β. Membranes isolated from erythrocytes pretreated with 4β-phorbol-12β-myristate-13α-acetate (PMA) exhibited a decreased capacity for Gα11-mediated activation of purified, reconstituted PLC-β1. This inhibitory effect was dependent on both the time and concentration of PMA incubation and occurred as a decrease in the efficacy of GTPγS for activation of PLC-β1, 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 Gα11 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 Gα11 is the target for PKC and that PKC-promoted phosphorylation of this protein results in a phosphorylation-dependent suppression of Gα11-mediated PLC-β1 activation.
Footnotes
- Received January 15, 1999.
- Accepted April 26, 1999.
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Send reprint requests to: Dr. Michelle L. Cunningham, CB# 7365, Mary Ellen Jones Building, University of North Carolina School of Medicine, Chapel Hill, NC 27599. E-mail:cunnml{at}med.unc.edu
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↵1 Current address: College of Pharmacy, 203 Pharmacy Building, Oregon State University, Corvallis, OR 97331.
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This work was supported by U.S. Public Health Service Grant GM-29536, a National Research Service Award to T.M.F., and a Howard Hughes Medical Institute Predoctoral Fellowship to M.L.C.
- The American Society for Pharmacology and Experimental Therapeutics
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