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 32P 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.
Bondzi
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Department of Microbiology/Immunology, Medical College of Virginia of Virginia Commonwealth University and McGuire VA Medical Center, Richmond, Virginia, VA 23249, USA.
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Krystal
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D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D018288:Carcinoma, Small Cell / Q000201:enzymology; D003720:Densitometry; D004790:Enzyme Induction; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000201:enzymology; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000276:immunology / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D020298:U937 Cells
D000906:Antibodies; D000972:Antineoplastic Agents, Phytogenic; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D017239:Paclitaxel