Transcriptional activation of immediate early genes by treatment of cells with interferons (IFNs) is essential for their antiviral and antiproliferative effects, properties of these cytokines which have allowed them to be therapeutically useful in the treatment of cancer. The primary signaling cascade responsible for the expression of interferon stimulated genes (ISGs) involves the activation of the Jak/Stat pathway. In addition to being tyrosine phosphorylated, several Stats (transcription factors) including Stat1alpha, Stat3 and Stat4 are phosphorylated on a conserved serine residue which is a consensus phosphorylation site for mitogen activated protein kinases (MAPK). Incubation of cells with either IFNalpha/beta or IFNgamma activates B- Raf and Raf-1, related serine kinases responsible for the ultimate activation of p42MAPK. Activation of Raf-1 requires both Jak1 and Stat1. In cells derived from mouse embryos with a targeted deletion of B-Raf, the antiproliferative actions of IFNalpha are lost and the antiviral effects of this cytokine are severely impaired as well as IFN activation of Stat1. We will test the hypothesis that B-Raf and/or the substrates which it regulates are essential signaling components for the antiviral and antigrowth effects of IFNalpha. We will test this hypothesis by performing the following Specific Aims: 1a) Determine whether activation of IFNalpha stimulated formation of ISGF3, and interferon stimulated gene expression are also diminished in B-Raf-null ES cells. 1b) Determine whether activation of B-Raf requires Jak1 and Stat1. 1c) Identify domains in Jak1 and Stat1 involved in binding and/or activation of B-Raf. 2a) Determine whether these domains inhibit IFN stimulated B-Raf and MAPK activities, and the antiviral or antiproliferative actions of these cytokines when expressed in cultured cells. 2b) Determine the domains in B-Raf which are required for this protein to permit IFN stimulated tyrosine phosphorylation of the Stat proteins and allow for the antigrowth and antiviral activities of tyrosine phosphorylation of the Stat proteins and allow for the antigrowth and antiviral activities of IFNalpha in B-Raf-null ES cells. 3) Characterize and Purify Novel Proteins which may be required for IFN activation of B-Raf kinase.
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