Identification of ImmediateEarly Gene Targets of the Raf-1 Serine/Threonine Protein Kinase Using an Estradiol-Dependent Fusion Protein, Raf-1:ER

The Raf-1 serine/threonine protein kinase is a central component of the Ras/Raf/MEK/MAP kinase cascade, a highly conserved signaling pathway responsible for the transmission of signals emanating from cell surface growth factor, cytokine, and hormone receptors into the nucleus (1 –4 ). Receptor-dependent activation of the Ras GTPase results in recruitment of Raf-1 to the plasma membrane whereupon Raf-1 kinase is activated by a mechanism that is not yet fully elucidated. Subsequently, Raf-1 phosphorylates and activates the dual specificity kinase, map kinase kinase (MEK/MKK), which in turn phosphorylates and activates the mitogen activated protein (MAP/ERK) kinases. MAP kinases then translocate to the nucleus, where they effect the phosphorylation of a variety of transcription factors, thereby evoking changes in gene expression. Much interest has focused on the Ras/Raf/MEK/MAP kinase cascade in view of its important role in regulating cell growth and differentiation in response to polypeptide growth factors and also because constitutive activation of this pathway at level of Ras, Raf, or MEK can promote oncogenic transformation of mammalian cells (2 ,3 ). Since it is widely perceived that phenotypic responses to Ras/Raf/MEK/MAP kinase signaling involve changes in gene expression, we have employed a conditional form of the Raf-1 protein kinase (5 ) in conjunction with differential display PCR (6 ,7 ) to identify immediate-early target genes of Raf-1 signaling.