OHNO Shigeo Yokohama City University School of Medicine, Professor, 医学部・第二生化学教室, 教授 (10142027)
YAJIMA Yukiko The Tokyo Metropolitan Inst. of Med. Sci., Tumor Cell Biology, Research Scientis, 腫瘍細胞研究部門, 主任研究員 (60090114)
|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1990 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Protein kinase C (PKC) is a major component of the transmembrane signaling system. Recently, we have discovered a novel protein kinase C isozyme, nPKCepsilon, and found that nPKCepsilon, is distinguished structurally and biochemically from the conventional PKCs (cPKCalpha, betaI/II, and gamma) in the rack of the C2 sequence and Ca^<2+>-independencies for membrane association, phorbol ester binding, and kinase activity.
In this study, we have examined the physiological role of PKC isozymes in rat pituitary GH cells. nPKCepsilon was identified together with two conventional Ca^<2+>-dependent PKCs, PKCalpha and beta II. These PKCs are down-regulated differently when cells are stimulated by extracellular stimuli ; phorbol esters deplete PKCbeta II and nPKCepsilon from the cells more rapidly than PKCalpha, whereas thyrotropin-releasing hormone (TRH) depletes nPKCepsilon exclusively. However, translocation of PKC alpha and beta II to the membranes is elicited by both TRH and phorbol esters. These results suggest that TRH and phorbol ester activate PKCalpha and beta II differently but nPKCepsilon is stimulated similarly by both stimuli.
Subsequently, we found that TRH-stimulated prolactin (PRL) secretion (30 min) was significantly retained in the Ca^<2+>-depleted GH cells by pretreatment with EGTA and arachidonic acid. This Ca^<2+>-independent PRL secretion was almost completely suppressed by a potent inhibitor of PKC isozymes including NPKCS, H7. Moreover, in the cells down-regulated for cPKCbeta II and nPKCepsilon by pretreatment with phorbol esters, the secretion was abolished. These findings suggest that nPKCepsilon may be involved in the Ca^<2+>-independent secretory process.
We believe that the present study would facilitate the understanding of the cellular signaling pathways involving cPKCs and nPKCs.