|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1998 : ¥1,900,000 (Direct Cost : ¥1,900,000)
A protein kinase C isozyme, nPKCε plays a key role in prolactin (PRL) secretion upon the stimulation of thyrotropin-releasing hormone (TRH). Here we examined the downstream mechanism following nPKCε activation by two-dimensional gel electrophoresis. Exposure of rat pituitary GHィイD24ィエD2CィイD21ィエD2 cells to TRH causes an immediate stimulation of the phosphorylation of six proteins, a p80 (MィイD2rィエD2 〜 80,000; PI 〜 4.3), three p52 (p52a, b and c; MィイD2rィエD2 〜 52,000; pI 〜5.75, 5.7, and 5.6, respectively), and two p19 (p19a and b: MィイD2rィエD2 〜19,000; pI 〜5.6 and 5.5, respectively). Phorbol ester, a potent activator of PKC, also enhances these phosphorylations, whereas bisindolylmaleimide, a specific inhibitor of PKC, clearly inhibits the phosphorylation of p80 and p52. p80, p.52, and p19were identified as myristoylated alanine-rich C kinase substrate (MARCKS), keratin (K), and sathmin, respectively, as assessed by their 2D-gel electrophoretic profiles and their biochemical properties. In nPKCε-overexpressing clones, the phosphorylated levels of MARCKS and K but not stathmin were appreciably high in the resting state, and additionally enhanced and sustained upon TRH stimulation, correlating with the enhanced activation of overexpressing nPKCε. TRH stimulates the rapid release of MARCKS and K, regulatory components in two independent cytoskeletal architectures, micro- and intermediate-filaments, respectively, are the major substrates of nPKCε in vivo, and that these phosphorylations may regulate the hormonal secretion.