Elevation of cytosolic Ca2+ by Alpha-1-adrenergic agents and vasopressin in liver, leading to increased glycogenolysis, is associated with receptor-mediated hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the intracellular messengers, inositol 1,4,5 trisphosphate (IP3) and 1,2-diacylglycerol. IP3 mobilizes Ca2+ from intracellular stores, and diacylglycerol, through a phospholipid-dependent protein kinase (protein kinase C) can effect changes in protein phosphorylation. The overall objective is to assess the significance of protein kinase C in the actions of Ca2+-mobilizing hormones, and potential cross-relationships with the actions of glucagon and insulin. These studies will focus in particular on the mechanism whereby the tumor-promoting phorbol ester (PMA), which can directly activate protein kinase C, inhibits Alpha 1-agonist-induced Ca2+ mobilization, but has little effect on that induced by vasopressin. The main hypothesis to be tested is that phosphorylation by protein kinase C of a particular protein, possibly closely associated with the Alpha-1-receptor, inhibits the signal transduction process. The particular aims addressed are: (1) Delineation of the inhibitory site of action of PMA on Alpha-1-agonist-induced Ca2+ mobilization; (2) Characterization of protein kinase C and its substrate proteins in liver; (3) Identification of the phosphoprotein(s) responsible for Alpha-1-receptor down-regulation; (4) Determination of changes in hepatocyte 32-P-protein labeling by insulin and possible relationships with PMA-induced phosphorylation. The locus of the PMA effect on Alpha-1-events will be addressed by examining effects of PMA wiht or without exogenous protein kinase C on [3H] prazosin binding, and on breakdown of [32P] PIP2 in membranes prepared from hepatocytes prelabeled with 32P. The five proteins thus far identified as protein kinase C substrates (Mr values of 68,000, 52,000, 36,000 and two 16,000 dalton proteins) will be further characterized and the regulatory properties of purified liver protein kinase C will be examined. Insulin-induced changes in 32P hepatocyte protein labeling will be examined by two-dimensional SDS-PAGE. This study should provide new information regarding early events associated with the actions of a tumor-promoting agent, clarify the mechanism of receptor-mediated inositol lipid breakdown and indicate whether the inhibitory effects of insulin and PMA on Alpha-1-adrenergic actions occur through a similar mechanism, involving phosphorylation of a key regulatory protein albeit mediated by different protein kinases.
| Beeler, J F; Cooper, R H (1995) Regulation of hepatocyte plasma membrane alpha 1-adrenergic receptors by 4 beta-phorbol 12-myristate 13-acetate. Biochem J 305 ( Pt 1):73-9 |
| Beeler, J F; Cooper, R H (1993) Rapid and reversible uncoupling of the hepatic alpha 1-adrenergic receptor and guanine-nucleotide-binding protein by phorbol 12,13-dibutyrate. Biochim Biophys Acta 1176:339-42 |
| Chuang, L F; Cooper, R H; Yau, P et al. (1987) Protein kinase C phosphorylates leukemia RNA polymerase II. Biochem Biophys Res Commun 145:1376-83 |
