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

被引:24
作者
Bondzi, C
Grant, S
Krystal, GW
机构
[1] McGuire VA Med Ctr, Richmond, VA 23249 USA
[2] Virginia Commonwealth Univ, Med Coll Virginia, Dept Microbiol Immunol, Richmond, VA 23249 USA
[3] Virginia Commonwealth Univ, Med Coll Virginia, Dept Med, Richmond, VA 23249 USA
关键词
Raf-1; activation-specific antibody; kinase assay;
D O I
10.1038/sj.onc.1203862
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:5030 / 5033
页数:4
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