Protein Kinase C-Promoted Inhibition of G a 11-Stimulated Phospholipase Cb 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 Ga11, independent of potential effects on the receptor or on PLC-b. Membranes isolated from erythrocytes pretreated with 4b-phorbol-12b-myristate-13a-acetate (PMA) exhibited a decreased capacity for Ga11-mediated activation of purified, reconstituted PLC-b1. This inhibitory effect was dependent on both the time and concentration of PMA incubation and occurred as a decrease in the efficacy of GTPgS for activation of PLC-b1, 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 Ga11 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 Ga11 is the target for PKC and that PKCpromoted phosphorylation of this protein results in a phosphorylation-dependent suppression of Ga11-mediated PLC-b1 activation. Various receptors transduce signals through heterotrimeric G proteins of the Gq family, resulting in activation of phospholipase C (PLC)-b isoenzymes and subsequent cleavage of membrane phosphatidylinositol(4,5)P2 [PtdIns(4,5)P2] to the second messengers inositol(1,4,5)P3 [Ins(1,4,5)P3] and diacylglycerol (Berridge and Irvine, 1987). Ins(1,4,5)P3 initiates release of calcium from intracellular stores, and diacylglycerol, in conjunction with calcium and phospholipids, activates protein kinase C (PKC). Agonist-induced desensitization is an important regulatory process in inositol lipid signaling (Fisher, 1995), although the precise mechanisms involved are unclear. Receptor-promoted desensitization is mimicked in several cell types by activation of protein kinase C (Orellana et al., 1985; Rittenhouse and Sasson, 1985) and is prevented by downregulation of PKC by chronic exposure to 4b-phorbol-12bmyristate-13a-acetate (PMA; Hepler et al., 1988) or by PKC inhibitors (Galas and Harden, 1995). These observations strongly suggest that PKC plays a negative regulatory role in phosphoinositide hydrolysis, probably by causing a phosphorylation-dependent change in the activity of another component of the pathway. Potential targets of PKC-catalyzed phosphorylation in this putative negative-feedback system include the receptor, the G protein, the effector enzyme PLC-b, or other less well-defined membrane-signaling proteins. GTPgS-stimulated PLC-b activity is attenuated in membranes isolated from PMA-treated cells (Orellana et al., 1987), suggesting that PKC acts on a protein downstream of