Role of protein kinases in the prolactin-induced intracellular calcium rise in Chinese hamster ovary cells expressing the prolactin receptor

There is still only limited understanding of the early steps of prolactin s ignal transduction in target cells. It has been shown that prolactin action s are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Ch inese hamster ovary (CHO) cells, stably transfected with the long form of r abbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R sig nal transduction studies. Spectrofluorimetric techniques were used to measu re intracellular calcium ([Ca2+](i)) in cell populations with Indo1 as a ca lcium fluorescent probe. We demonstrate that, although protein kinase C act ivation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induc ed PKC activation was not responsible for the early effect of prolactin on [Ca2+](i). Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+](i) and inhibition of PKA pathway did not affect the prolacti n response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+](i) (influx and release). W7, a ca lmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62) , only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerni ng the involvement of protein phosphorylations in the early effects of prol actin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphoryl ation is a crucial, very early step, since it controls K+ channel activatio n, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx.