Cook S J, Beltman J, Cadwallader K A, McMahon M, McCormick F
ONYX Pharmaceuticals, Inc., Richmond, California 94806, USA.
J Biol Chem. 1997 May 16;272(20):13309-19. doi: 10.1074/jbc.272.20.13309.
Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had little effect on the early peak of ERK1 activity but potentiated the sustained phase. Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing DeltaRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1. Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selective inhibitor of Gi-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by DeltaRaf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1DeltaRaf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression.
用溶血磷脂酸（LPA）或表皮生长因子（EGF）刺激大鼠1细胞会导致细胞外信号调节激酶1（ERK1）出现双相、持续激活。用放线菌酮或原钒酸钠预处理大鼠1细胞对ERK1活性的早期峰值影响不大，但会增强其持续阶段。放线菌酮也能增强表达DeltaRaf-1:ER（一种雌激素调节的致癌性人类Raf-1形式）的大鼠1细胞中的ERK1激活。由于放线菌酮不会增强MEK活性，但会消除通常在对EGF和LPA反应中出现的丝裂原活化蛋白激酶磷酸酶（MKP-1）的表达，我们推测MKP-1的表达水平可能是大鼠1细胞中ERK1活性的重要调节因子。用PD98059和百日咳毒素（一种Gi蛋白偶联信号通路的选择性抑制剂）抑制LPA刺激的MEK和ERK激活，仅使LPA刺激的MKP-1表达降低50%，这表明存在其他不依赖MEK和ERK的MKP-1表达途径。DeltaRaf-1:ER对MEK/ERK途径的特异性激活对MKP-1表达几乎没有影响，这表明Raf/MEK/ERK途径的激活对于大鼠1细胞中MKP-1的表达是必要的，但并不充分。蛋白激酶C（PKC）的激活在生长因子刺激的MKP-1表达中作用不大，但LPA和EGF诱导的MKP-1表达被缓冲细胞内钙离子浓度（[Ca2+]i）所阻断，导致ERK1激活的持续阶段增强，而不增强MEK活性。在大鼠1DeltaRaf-1:ER细胞中，当在离子霉素、佛波醇12-肉豆蔻酸酯13-乙酸酯或冈田酸存在的情况下用雌激素刺激细胞时，我们观察到MKP-1表达有很强的协同作用，而在这些试剂对ERK激活没有协同作用的条件下。这些结果表明，Raf/MEK/ERK途径的激活不足以诱导MKP-1的表达，而是需要其他信号，如Ca2+，来完全重建生长因子所引发的反应。通过这种方式，ERK依赖和不依赖的信号可能调节MKP-1的表达、ERK1持续活性的大小，进而调节基因表达。
