Raf and fibroblast growth factor phosphorylate Elk1 and activate the serum response element of the immediate early gene pip92 by mitogen-activated protein kinase-independent as well as -dependent signaling pathways

Previous studies have shown that a mitogen activated protein (MAP) kinase (MEK)-independent signaling pathway is required by activated Raf or fibroblast-derived growth factor (FGF) for the differentiation of rat hippocampal neuronal H19-7 cells. We now demonstrate that both Raf and FGF similarly induce prolonged transcription and translation of the immediate early gene pip92 in the absence of activation of the MAP kinases (MAPKs) ERK1 and ERK2. To determine the mechanism by which this occurs and to identify novel Raf-activated signaling pathways, we investigated the induction of the pip92 promoter by both FGF and an estradiol-activated Raf-1-estrogen receptor fusion protein (ΔRaf-1:ER) in H19-7 cells. Deletion analysis of the pip92 promoter indicated that activation by the MAPK-independent pathway occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE by using a heterologous thymidine kinase promoter showed that both an Ets and CArG-like site are required. Elk1, which binds to the Ets site, was phosphorylated both in vitro and in vivo by the MAPK- independent pathway, and phosphorylation of an Elk1-GAL4 fusion protein by this pathway was sufficient for transactivation. Finally, at least two Elk1 kinases were fractionated by gel filtration, and analysis by an in-gel kinase assay revealed at least three novel Raf-activated Elk1 kinases. These results indicate that both FGF and Raf activate MAPK-independent kinases that can stimulate Elk1 phosphorylation and immediate early gene transcription.