Detection of Activated Signaling Intermediates

Site specific phosphorylation represents a major pathway through which signal transductionstimuli influence gene expression. Phosphorylation of critical sites can influence binding of transcriptionfactors to specific regulatory elements, affect heterodimer formation between different members withinbroad families of transcription factors and influence the effect of transcription factor binding on geneexpression. As a prototype system for the generation of phosphorylation site specific anti-bodies to keyregulatory mammalian transcription factors we propose the development, in Phase I, of affinity-purifiedrabbit polyclonal antibodies to peptides encompassing phosphorylated and non-phosphorylated c-jun p39at serine residues 63,73 and 246. These will be assayed for ability to discriminate phosphorylated andnon-phosphorylated forms of purified c-jun p.39. In parallel, agarose conjugated antibodies andconsensus sequence oligonucleotides will be developed as solid phase reagents for enrichment of c-junp39 form nuclear extracts to enhance analysis of its extent of phosphorylation. In Phase II and III, thesestudies will be extended to the development of c-jun p39 phosphorylation site specific monoclonalantibodies, analysis of phosphorylation sites on transcription factors which form heterodimericcomplexes with jun, such as members of the CREB/AFT family, and the isolation of hybridoma clonesproducing monoclonal antibodies specific for the phosphorylated form of the dipeptide Ser-Pro.