Burgering B M, Pronk G J, van Weeren P C, Chardin P, Bos J L
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
EMBO J. 1993 Nov;12(11):4211-20. doi: 10.1002/j.1460-2075.1993.tb06105.x.
In fibroblasts, stimulation of receptor tyrosine kinases results in the activation of the extracellular signal-regulated kinase 2 (ERK2). The major signalling pathway employed by these receptors involves the activation of p21ras and raf-1 kinase. Here we show that in NIH3T3 and rat-1 fibroblasts, elevation of the intracellular cAMP level results in the inhibition of ERK2 activation induced by PDGF, EGF and insulin treatment. Analysis of various signalling intermediates shows that cAMP interferes at a site downstream of p21ras, but upstream of raf-1 kinase. Inhibition by cAMP depends on both the cAMP concentration and the absolute amount of p21ras molecules bound to GTP, suggesting a mechanism of competitive inhibition. Also TPA-induced, p21ras-independent, activation of raf-1 kinase and ERK2 is inhibited by cAMP. We have used the inhibitory effect of cAMP to investigate whether phosphorylation of mSos, a p21ras nucleotide exchange factor, is dependent on the activity of the raf-1 kinase/ERK2 pathway. We found that phosphorylation of mSos, as monitored by a mobility shift, is delayed with respect to p21ras and ERK2 activation and is inhibited by cAMP in a similar cell type- and concentration-dependent manner as the inactivation of ERK2. These results provide evidence for a model of p21ras-directed signalling towards ERK2 that feeds back on mSos by regulating its phosphorylation status and that can be negatively modulated by protein kinase A and positively modulated by protein kinase C action.
在成纤维细胞中，受体酪氨酸激酶的刺激会导致细胞外信号调节激酶2（ERK2）的激活。这些受体所采用的主要信号通路涉及p21ras和raf-1激酶的激活。在此我们表明，在NIH3T3和大鼠-1成纤维细胞中，细胞内cAMP水平的升高会抑制由血小板衍生生长因子（PDGF）、表皮生长因子（EGF）和胰岛素处理诱导的ERK2激活。对各种信号中间体的分析表明，cAMP在p21ras的下游、但raf-1激酶的上游位点产生干扰。cAMP的抑制作用取决于cAMP浓度以及与GTP结合的p21ras分子的绝对量，提示存在一种竞争性抑制机制。此外，佛波酯（TPA）诱导的、不依赖p21ras的raf-1激酶和ERK2激活也受到cAMP的抑制。我们利用cAMP的抑制作用来研究p21ras核苷酸交换因子mSos的磷酸化是否依赖于raf-1激酶/ERK2途径的活性。我们发现，通过迁移率变动监测到的mSos磷酸化相对于p21ras和ERK2激活有所延迟，并且在与ERK2失活类似的细胞类型和浓度依赖性方式下受到cAMP的抑制。这些结果为一种由p21ras导向ERK2的信号传导模型提供了证据，该模型通过调节mSos的磷酸化状态对其产生反馈作用，并且可被蛋白激酶A负向调节，被蛋白激酶C作用正向调节。
