Gause K C, Homma M K, Licciardi K A, Seger R, Ahn N G, Peterson M J, Krebs E G, Meier K E
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425.
J Biol Chem. 1993 Aug 5;268(22):16124-9.
Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.
佛波酯敏感型和耐药型EL4胸腺瘤细胞系在对佛波酯作出反应时激活丝裂原活化蛋白激酶（MAPK）的能力有所不同。用佛波酯处理野生型EL4细胞会导致MAPK和pp90rsk激酶（一种MAPK的底物）迅速激活，而在变异型EL4细胞中，这两种激酶均不会因佛波酯而被激活。本研究检测了野生型和变异型EL4细胞中MAPK激酶（MAPKK，一种MAPK的激活剂）的激活情况。在使用从经佛波酯处理的野生型EL4细胞制备的胞质提取物或Mono Q柱级分进行体外磷酸化反应后，观察到一种被鉴定为MAPK的40 kDa底物发生了磷酸化。在Mono Q阴离子交换色谱中，MAPKK活性与一部分无活性的MAPK共同洗脱，从而能够检测同时含有这两种激酶的级分中的MAPKK活性。这种MAPKK活性存在于经佛波酯处理的野生型细胞中，但不存在于经佛波酯处理的变异型细胞中，也不存在于未处理的野生型或变异型细胞中。野生型细胞的MAPKK能够激活从野生型或变异型细胞制备的MAPK。用冈田酸处理细胞后，野生型和变异型EL4细胞中的MAPKK活性均可被刺激。这些结果表明，变异型EL4细胞无法因佛波酯而激活MAP激酶是由于未能激活MAPKK。因此，赋予变异型EL4细胞佛波酯抗性的步骤位于蛋白激酶C的激活与MAPKK的激活之间。
