Probing structure and function of the raf protein kinase domain with monoclonal antibodies.

Five monoclonal antibodies were generated against the raf kinase domain. All antibodies react with different isozymes of the raf family, as well as Raf proteins from different species, albeit with differential affinities. Epitope mapping showed all five epitopes clustered in the vicinity of the conserved APE sequence. Although thought to be an essential part of the catalytic site, antibody binding to that domain does not affect kinase activity in vitro or the capability to specifically associate with other cellular proteins. Based on a detailed dissection of the epitopes, a comparative analysis of secondary structure predictions indicates a common structural motif in that region, which is highly conserved amongst protein kinases of the serine/threonine as well as the tyrosine class.