A novel assay for the measurement of Raf-1 kinase activity

Raf-1 is a serine-threonine protein kinase that functions as a central component of the mitogen-activated protein kinase signal transduction pathway, Raf-1 activity is currently assayed in vitro by either measuring P-32 incorporation into MEK, Raf-1's only characterized substrate, or by using the phosphorylated MEK to initiate a coupled assay culminating in the phosphorylation of myelin basic protein by MAP kinase, These assays are plagued by a potential lack of specificity in the case of the former, and the time consuming and error-prone nature of the later indirect assay, In this report, we demonstrate a novel single step assay for Raf-1 kinase activity based on phosphorylation of recombinant MEK-1, detected using an activation-specific MEK antibody that recognizes MEK only when specifically phosphorylated by Raf-1 on Ser 217 and Ser 221, The assay readily detected stem cell factor-mediated Raf-1 activation, MEK phosphorylation by immunoprecipitated Raf-1 plateaued at 10 min following initiation of the kinase reaction and was completely dependent on the inclusion of Raf-1 There was a linear correlation between the degree of MEK phosphorylation and the amount of Raf-1 protein immunoprecipitated, In addition to detecting growth factor-mediated activation, the assay was also able to detect paclitaxel-mediated Raf-1 activation, This assay is rapid, sensitive, and specific and therefore is a marked improvement over currently utilized techniques.