We have shown previously that novel protein kinase C epsilon (nPKC epsilon) plays a key role in the basal and thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion in rat pituitary GH(4)C(1) cells (Akita et al., J. Biol. Chem. 1994, 269, 4653-4660). Here we examined the region downstream of nPKC epsilon activation in order to understand the molecular mechanism by which nPKC epsilon mediates TRH-induced signal transduction. Exposure of GH(4)C(1) cells to TRH causes a stimulation of the phosphorylation of a p80 (Mr similar to 80 000, pl similar to 4.3) and two p19 (p19a and b; M-r similar to 19 000, pl similar to 5.6 and 5.5, respectively). Phorbol ester, a potent activator of protein kinase C (PKC), also enhances these phosphorylations, whereas bisindolylmaleimide I, a specific inhibitor of PKC, clearly inhibits the phosphorylation of p80. p80 and p19 were identified as myristoylated alanine-rich C kinase substrate (MARCKS) and stathmin, respectively, as assessed by their two-dimensional gel electrophoretic profiles and their stabilities to heat and acid treatment. in nPKC epsilon-overexpressing stable clones, the phosphorylated level of MARCKS but not stathmin was high in the resting state, and enhanced and sustained upon TRH stimulation, correlating with the increased activation of nPKC epsilon. TRH stimulates the release of MARCKS from the membrane/cytoskeletal fraction to the cytosol fraction. These results, taken together with previous data concerning PRL secretion, suggest that MARCKS, a regulatory component of the cytoskeletal architecture, is the major substrate of nPKC epsilon in vivo, and that its phosphorylation may regulate TRH-stimulated PRL secretion.