Estrogens and growth factors such as epidermal growth factor (EGF) act as mitogens promoting cellular proliferation in the breast and in the reproductive tract. Although it was considered originally that these agents manifested their mitogenic actions through separate pathways, there is a growing body of evidence suggesting that the EGF and estrogen-mediated signaling pathways are intertwined. Indeed, it has been demonstrated recently that 17␤-estradiol (E2) can induce a rapid activation of mitogenactivated protein kinase (MAPK) in mammalian cells, an event that is independent of both transcription and protein synthesis. In this study, we have used a pharmacological approach to dissect this novel pathway in MCF-7 breast cancer cells and have determined that in the presence of endogenous estrogen receptor, activation of MAPK by E2 is preceded by a rapid increase in cytosolic calcium. The involvement of intracellular calcium in this process was supported by the finding that the presence of EGTA and Ca 2؉ -free medium did not affect the activation of MAPK by E2 and, additionally, that this response was blocked by the addition of the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetate. Cumulatively, these data indicate that the estrogen receptor, in addition to functioning as a transcription factor, is also involved, through a nongenomic mechanism, in the regulation of both intracellular calcium homeostasis and MAPK-signaling pathways. Although nongenomic actions of estrogens have been suggested by numerous studies in the past, the ability to link estradiol and the estrogen receptor to a well defined signaling pathway strongly supports a physiological role for this activity.

Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R143, K144, and K148 appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.