PROLACTIN INHIBITS EPIDERMAL GROWTH FACTOR-INDUCED RAS-MAPK SIGNALINGIN MAMMARY EPITHELIAL-CELLS

Previously, our laboratory has shown that prolactin (PRL) inhibits epi dermal growth factor (EGF)-induced DNA synthesis. One pathway for the initiation of DNA synthesis is EGF-receptor (EGF-R) signaling through Ras and mitogen-activated protein kinase (MAPK). To determine the effe cts of PRL on EGF-induced MAPK activation and phosphorylation, MAPK or phosphotyrosine (Tyr(P)) was immunoprecipitated from normal murine ma mmary epithelial (NMuMG) cells treated with PRL (100 ng/ml) and/or EGF (10 ng/ml) for 10-min periods. EGF-induced phosphorylation and activa tion were then examined by Western analysis and a myelin basic protein (MBP)-specific kinase assay. The p42 isoform of MAPK showed a distinc t decrease in activity and phosphorylation when cells were treated wit h PRL. Concluding that PRL affects EGF signaling upstream of MAPK, we examined the effect of PRL on EGF-induced Ras activity. NMuMG cells we re incubated with [P-32]orthophosphoric acid, treated as described abo ve, immunoprecipitated with an antibody specific to Ras, and nucleotid es were eluted and separated by TLC. Ras activity as measured by GTP:G DP ratio was increased by EGF, but not by PRL. Additionally, PRL, in c ombination with EGF abolished the ability of EGF to induce Ras activit y, Those studies suggest that PRL alters the EGF signaling pathway ups tream of Ras. Because Ras activation by EGF involves EGF-stimulated as sociation of EGF-R with Grb2, the EGF-R was immunoprecipitated and a W estern blot was probed for Grb2. As expected we found that EGF stimula ted an association of EGF-R with Grb2, PRL, however, blocked this asso ciation. When we looked at the ability of Shc to associate with the EG F-R, we found that PRL and EGF had little effect on this association. The studies demonstrate that PRL either directly or indirectly inhibit s the ability of EGF to induce EGF-R association with Grb2, to activat e Ras, and to activate and phosphorylate MAPK.