Kinetic and biochemical correlation between sustained p44ERK1 (44 kDa extracellular signal-regulated kinase 1) activation and lysophosphatidic acid-stimulated DNA synthesis in Rat-1 cells
Kinetic and biochemical correlation between sustained p44ERK1 (44 kDa extracellular signal-regulated kinase 1) activation and lysophosphatidic acid-stimulated DNA synthesis in Rat-1 cells
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DOI：
10.1042/bj3200237
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发表时间：
1996-11-15
影响因子：
4.1
通讯作者：
McCormick, F
中科院分区：
文献类型：
--
作者：
Cook, SJ;McCormick, F
利用大鼠-1成纤维细胞研究细胞外信号调节激酶1 （ERK1）持续激活在溶血磷脂酸（LPA）刺激的有丝分裂信号传导中的作用。与血清一样，有丝分裂剂量的LPA刺激双期、持续的ERK激活，并持续到G1/S边界。lpa刺激的ERK在10分钟（反应峰时间）后激活的EC（50）比持续反应3小时后的EC（50）或DNA合成后的EC（50）左2个数量级，结果是非有丝分裂剂量刺激了最大峰反应，但没有第二阶段反应。为了补充这些研究，我们使用定义的生化抑制剂和模拟物检查了不同信号通路在调节ERK激活的持续和急性期中的作用。蛋白激酶C和Ca2+通量的激活在LPA对大鼠1细胞ERK1活性的调节中发挥了次要和短暂的作用。LPA刺激的ERK1持续激活被百日咳毒素完全抑制，而早期峰值反应仅部分受到影响；这与百日咳毒素对lpa刺激的DNA合成的特异性抑制有关。选择性酪氨酸激酶抑制剂herbimycin A完全抑制LPA持续的ERK1激活，但同样，早期反应仅部分被抑制。此外，低剂量的司陶孢素可抑制LPA对ERK1的激活。herbimycin A和staurosporine对LPA的反应是选择性的，但对表皮生长因子的反应没有影响。结果表明，在lpa刺激的大鼠1细胞中，持续的ERK1激活与DNA合成之间存在很强的相关性。此外，LPA激活ERK的两个离散阶段受到至少两种不同信号通路的组合调节；鼠-1细胞中ERK1的持续激活是通过G(i)-或G(o)-介导的途径进行的，该途径也可能涉及酪氨酸激酶。
Rat-1 fibroblasts were used to study the role of the sustained activation of extracellular signal-regulated kinase 1 (ERK1) in lysophosphatidic acid (LPA)-stimulated mitogenic signalling. Mitogenic doses of LPA, like serum, stimulated biphasic, sustained, ERK activation that persisted towards the G1/S boundary. The EC(50) for LPA-stimulated ERK activation after 10 min, the time of peak response, was 2 orders of magnitude to the left of that for the sustained response after 3 h or that for DNA synthesis after 22 h, with the result that non-mitogenic doses stimulated a maximal peak response but no second phase. To complement these studies, we examined the role of different signal pathways in regulating the sustained and acute phases of ERK activation using defined biochemical inhibitors and mimetics. Activation of protein kinase C and Ca2+ fluxes played a minor and transient role in regulation of ERK1 activity by LPA in Rat-1 cells. Sustained ERK1 activation stimulated by LPA was completely inhibited by pertussis toxin, whereas the early peak response was only partly affected; this is correlated with the specific inhibition of LPA-stimulated DNA synthesis by pertussis toxin. The selective tyrosine kinase inhibitor herbimycin A completely inhibited sustained ERK1 activation by LPA but, again, the early phase of the response was only partially inhibited. In addition, low doses of staurosporine inhibited ERK1 activation by LPA. The effects of herbimycin A and staurosporine were selective for the response to LPA but did not affect that to epidermal growth factor. The results suggest a strong correlation between sustained ERK1 activation and DNA synthesis in LPA-stimulated Rat-1 cells. Furthermore, the two discrete phases of ERK activation by LPA are regulated by a combination of at least two different signalling pathways; the sustained activation of ERK1 in Rat-1 cells proceeds via a G(i)- or G(o)-mediated pathway which may also involve a tyrosine kinase.