OVERPRODUCTION OF A CA2-INDEPENDENT PROTEIN-KINASE-C ISOZYME, NPKC-EPSILON, INCREASES THE SECRETION OF PROLACTIN FROM THYROTROPIN-RELEASINGHORMONE-STIMULATED RAT PITUITARY GH(4)C(1) CELLS()

Rat pituitary GH(4)C(1) cells express protein kinase C (PKC) transcrip ts for cPKC alpha, cPKC beta II, nPKC delta, nPKC epsilon, nPKC eta, a nd aPKC xi, but not for cPKC gamma or nPKC theta. Of the transcripts p roduced, the nPKC epsilon isoform is the most abundant. Transfection o f GH(4)C(1) cells with an expression plasmid containing nPKC epsilon c DNA leads to the transient overexpression of cellular nPKC epsilon and confers enhanced phorbol ester binding activity. Transient expression of an inactive point mutant (nPKC epsilon(K-->R)) Of nPKC epsilon, wh ere Lys(436) at the putative ATP-binding site is replaced with Arg, al so confers elevated binding activity. However, only overproduction of the wild type in transfected cells increases the basal levels and stim ulates the secretion of prolactin (PRL) by 12-O-tetradecanoylphorbol-1 3-acetate or thyrotropin-releasing hormone (TRH). In stable clones ove rexpressing nPKC epsilon, immunocytofluorescence and immunoblot experi ments indicated that TRH causes the rapid translocation and down-regul ation of an appreciable fraction of nPKC epsilon. Both the basal and T RH-stimulated levels of PRL secretion are clearly correlated with the expression level of nPKC epsilon but not with the TRH receptor densiti es in these clones. The dose dependence of TRH-stimulated secretion we re similar in all cells overexpressing cPKC alpha, cPKC beta II, nPKC epsilon, and nPKC delta, but the enhancement of PRL secretion was spec ific for the overproduction of nPKC epsilon; no effect was found when other isozymes were overproduced. These findings clearly demonstrate t hat the expression level of nPKC epsilon in GH(4)C(1) cells is rate-li miting for basal and TRH-stimulated PRL secretion, and they provide th e first direct evidence that nPKC epsilon plays a key role in hormonal secretory processes.