title: Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells.
abstract: GH4C1 cells, which express Ca(2+)-dependent alpha- and beta- as well as Ca(2+)-independent gamma-, epsilon- and zeta-protein kinase C (PKC) isozymes, provide a cell culture model for studying isozyme-specific properties and functions. Hormonal activation of PKCs regulates the differentiated functions of these cells, namely secretion and synthesis of prolactin (PRL). We previously reported that thyrotropin-releasing hormone (TRH) selectively down-modulates epsilon-PKC with no effect on alpha- or beta-PKCs (Kiley, S.C., Schaap, D., Parker, P., Hsieh, L.-L., and Jaken, S. (1990) J. Biol. Chem. 265, 15704-15712). We now extend those studies to explore the relationship between TRH-stimulated diacylglycerol (DAG) levels and epsilon-PKC down-modulation. TRH stimulates three distinct DAG phases in GH cells. Phase 1 DAG peaks at 15 s, is accompanied by a 6-fold increase in intracellular Ca2+, and causes the redistribution of alpha-, beta-, delta, and epsilon-PKC isozymes from a soluble to a detergent-insoluble particulate compartment. Phase 2 DAG peaks at 10 min, is not associated with a Ca2+ signal, and does not activate PKC by any criteria tested. Phase 3 DAG peaks at 6 h and is sustained through 12 h. This novel DAG phase is not associated with increased intracellular Ca2+. The time course of phase 3 DAG formation corresponds to the time course of TRH-stimulated epsilon-PKC down-regulation; maximal effects are observed at 6-12 h for both events. Unlike alpha-, beta-, and delta-PKCs which are preferentially distributed in the soluble fraction of resting GH cells, epsilon-PKC is also distributed in the detergent-insoluble particulate fraction. The selective compartmentalization of epsilon-PKC in the particulate fraction may render this pool uniquely susceptible to proteolytic degradation. The time course of phase 3 DAG formation and epsilon-PKC down-modulation corresponds to the time course of decreasing PRL message synthesis in GH4 cells. The data suggests that loss of epsilon-PKC may be associated with the down-regulation of prolactin synthesis and that regulation of PRL gene transcription may be an epsilon-PKC-specific function in GH cells.
authors.affiliation: W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946-1099.
authors.forename: S C
authors.initials: SC
authors.lastname: Kiley
authors.forename: P J
authors.initials: PJ
authors.lastname: Parker
authors.forename: D
authors.initials: D
authors.lastname: Fabbro
authors.forename: S
authors.initials: S
authors.lastname: Jaken
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004075:Diglycerides / Q000302:isolation & purification / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D010911:Pituitary Neoplasms; D011493:Protein Kinase C / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
chemical_list: D004075:Diglycerides; D007527:Isoenzymes; D013973:Thyrotropin-Releasing Hormone; D011493:Protein Kinase C; D002118:Calcium