Ueda Y, Hirai S i, Osada S i, Suzuki A, Mizuno K, Ohno S
Department of Molecular Biology, Yokohama City University School of Medicine, 3-9, Fuku-ura, Kanazawa-ku, Yokohama 236, Japan.
J Biol Chem. 1996 Sep 20;271(38):23512-9. doi: 10.1074/jbc.271.38.23512.
Although the involvement of protein kinase C (PKC) in the activation of the mitogen-activated protein (MAP) kinase pathway has been implicated through experiments using 12-O-tetradecanoylphorbol-13-acetate (TPA), there has been no direct demonstration that PKC activates the MAP kinase pathway. A Raf-dependent intact cell assay system for monitoring the activation of MAPK/ERK kinase (MEK) and extracellular signal-related kinase (ERK) permitted us to evaluate the role of PKC isotypes in MAP kinase activation. Treatment of cells with TPA or epidermal growth factor resulted in the activation of MEK and ERK. The activation of the MAP kinase pathway triggered by epidermal growth factor was completely inhibited by dominant-negative Ras (RasN17), whereas the activation triggered by TPA was not, consistent with previous observations. The introduction of an activated point mutant of PKCdelta, but not PKCalpha or PKCepsilon, resulted in the activation of the MAP kinase pathway. The activation of MEK and ERK by an activated form of PKCdelta requires the presence of c-Raf and is independent of RasN17. These results demonstrate that activation of PKCdelta is sufficient for the activation of MEK and ERK and that the pathway operates in a manner dependent on c-Raf and independent of Ras.
尽管通过使用12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）的实验表明蛋白激酶C（PKC）参与了丝裂原活化蛋白（MAP）激酶途径的激活，但尚无直接证据表明PKC能激活MAP激酶途径。一种用于监测MAPK/ERK激酶（MEK）和细胞外信号相关激酶（ERK）激活的依赖Raf的完整细胞检测系统，使我们能够评估PKC同工型在MAP激酶激活中的作用。用TPA或表皮生长因子处理细胞会导致MEK和ERK的激活。表皮生长因子触发的MAP激酶途径的激活被显性负性Ras（RasN17）完全抑制，而TPA触发的激活则不受抑制，这与先前的观察结果一致。引入PKCdelta的活化点突变体可导致MAP激酶途径的激活，而PKCalpha或PKCepsilon则不能。活化形式的PKCdelta对MEK和ERK的激活需要c - Raf的存在，且不依赖于RasN17。这些结果表明，PKCdelta的激活足以激活MEK和ERK，且该途径以依赖c - Raf且不依赖Ras的方式运作。
