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()
Y. Akita et al., 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(), The Journal of biological chemistry, 269(6), 1994, pp. 4653-4660
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.