Involvement of protein kinase C ɛ in thyrotropin-releasing hormone-stimulated phosphorylation of the myristoylated alanine-rich C kinase substrate in rat pituitary clonal cells

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We have shown previously that novel protein kinase Cepsilon (nPKCepsilon) plays a key role in the basal and thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion in rat pituitary GH4C1 cells (Akita et al., J. Biol. Chem. 1994, 269, 4653-4660). Here we examined the region downstream of nPKCepsilon activation in order to understand the molecular mechanism by which nPKCepsilon mediates TRH-induced signal transduction. Exposure of GH4C1 cells to TRH causes a stimulation of the phosphorylation of a p80 (Mr approximately 80 000, pI approximately 4.3) and two p19 (p19a and b; Mr approximately 19 000, pI approximately 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 nPKCepsilon-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 nPKCepsilon. 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 nPKCepsilon in vivo, and that its phosphorylation may regulate TRH-stimulated PRL secretion.