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 KINASE-DEPENDENT SIGNALING PATHWAYS

Previous studies have shown that a mitogen activated protein (MAP) kin ase (MEK)-independent signaling pathway is required by activated naf o r fibroblast derived growth factor (FGF) for the differentiation of ra t hippocampal neuronal H19-7 cells. We now demonstrate that both Raf a nd FGF similarly induce prolonged transcription and translation of the immediate early gene pip92 in the absence of activation of the MAP ki nases (MAPKs) ERK1 and ERK2. To determine the mechanism by which this occurs and to identify mover RaF-activated signaling pathways, we inve stigated the induction of the pip92 promoter by both FGF and an estrad iol-activated Raf-1-estrogen receptor fusion protein (Delta Raf-1:ER) in H19-7 cells, Deletion analysis of the pip92 promotes indicated that activation by the MAPK-independent pathway occurs primarily within th e region containing a serum response element (SRE), Further analysis o f the SRE by using a heterologous thymidine kinase promoter showed tha t both an Ets and CArG-like site are required. Elk1, which hinds to th e Ets site, was phosphorylated both in vitro and in vivo by the MAPK-i ndependent 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 El k1 kinases. These results indicate that both FGF and Raf activate MAPK -independent kinases that ran stimulate Elk1 phosphorylation and immed iate early gene transcription.