Mallon R, Feldberg L R, Kim S C, Collins K, Wojciechowicz D, Hollander I, Kovacs E D, Kohler C
Oncology Research, Wyeth-Ayerst Research, 401 North Middletown Road, Pearl River, New York, 10965, USA.
Anal Biochem. 2001 Jul 1;294(1):48-54. doi: 10.1006/abio.2001.5151.
The Ras-MAPK signaling cascade transmits mitogenic stimuli from growth factor receptors and activated Ras to the cell nucleus. Inappropriate Ras activation is associated with approximately 30% of all human cancers. The kinase components of the Ras-MAPK signaling cascade are attractive targets for pharmaceutical intervention. Therefore, we have developed a high-throughput, nonradioactive ELISA method to monitor Raf and MEK1 kinase activity. In this assay system activated Raf phosphorylates and activates MEK1, which in turn phosphorylates MAPK. Antibodies that specifically detect phosphorylated MAPK (vs. nonphosphorylated MAPK) made enzyme-linked immunosorbent assay (ELISA) development possible. This assay detects inhibitors of Raf and/or MEK1 and has been used to screen large numbers of random compounds. The specific target of inhibition in the Raf/MEK1/MAPK ELISA can be subsequently identified by secondary assays which directly measure Raf phosphorylation of MEK1 or MEK1 phosphorylation of MAPK.
Ras-MAPK信号级联反应将有丝分裂刺激从生长因子受体和活化的Ras传递至细胞核。Ras的不适当激活与约30%的人类癌症相关。Ras-MAPK信号级联反应的激酶成分是药物干预的有吸引力的靶点。因此，我们开发了一种高通量、非放射性ELISA方法来监测Raf和MEK1激酶活性。在该检测系统中，活化的Raf磷酸化并激活MEK1，MEK1进而磷酸化MAPK。特异性检测磷酸化MAPK（相对于非磷酸化MAPK）的抗体使得酶联免疫吸附测定（ELISA）得以发展。该检测可检测Raf和/或MEK1的抑制剂，并已用于筛选大量随机化合物。Raf/MEK1/MAPK ELISA中抑制的具体靶点随后可通过直接测量MEK1的Raf磷酸化或MAPK的MEK1磷酸化的二次检测来确定。
