Tang Xiuwen, Wang Lijun, Proud Christopher G, Downes C Peter
Division of Cell Signalling, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Biochem J. 2003 Aug 15;374(Pt 1):137-43. doi: 10.1042/BJ20021910.
In 1321N1 astrocytoma cells, carbachol stimulation of M3 muscarinic cholinergic receptors, coupled to phospholipase C, evoked a persistent 10-20-fold activation of p70 S6 kinase (S6K1). This response was abolished by chelation of cytosolic Ca2+ and reproduced by the Ca2+ ionophore ionomycin, but was not prevented by down-regulation or inhibition of protein kinase C. Carbachol-stimulated activation and phosphorylation of S6K1 at Thr389 were prevented by rapamycin, an inhibitor of mTOR (mammalian target of rapamycin), or by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor. Carbachol also stimulated the phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), a second mTOR-dependent event, with similar potency to its effect on S6K1. This response was blocked by rapamycin, but was not markedly affected by 100 nM wortmannin, implying separate roles for mTOR and PI3K in S6K1 activation. Wortmannin abolished the carbachol-stimulated rise in PtdIns(3,4,5)P3 and greatly reduced unstimulated levels of this lipid. By contrast, an inhibitor of epidermal growth factor receptor kinase, AG1478, which prevents carbachol-stimulated ErbB3 transactivation, PI3K recruitment and protein kinase B activation in 1321N1 cells, reduced activation of S6K1 by no more than 30%. This effect was overcome by 10 nM insulin, which on its own did not stimulate S6K1, but increased cellular PtdIns(3,4,5)P3 concentrations comparably with carbachol alone. These observations distinguish obligatory roles for mTOR and PI3K in regulating S6K1, but imply that minimal PI3K activity is sufficient to permit stimulation of S6K1 by other activating factors such as increased cytosolic Ca2+ concentrations, which are essential to the muscarinic receptor-mediated response. Moreover, 4E-BP1 and hence, presumably, mTOR can be regulated independently of PI3K activation through these mechanisms.
在1321N1星形细胞瘤细胞中，卡巴胆碱刺激与磷脂酶C偶联的M3毒蕈碱胆碱能受体，可引起p70 S6激酶（S6K1）持续10至20倍的激活。这种反应可通过螯合胞质Ca2+而消除，并用Ca2+离子载体离子霉素重现，但蛋白激酶C的下调或抑制并不能阻止这种反应。雷帕霉素（一种mTOR（雷帕霉素的哺乳动物靶点）抑制剂）或渥曼青霉素（一种磷脂酰肌醇3激酶（PI3K）抑制剂）可阻止卡巴胆碱刺激的S6K1在Thr389位点的激活和磷酸化。卡巴胆碱还刺激了真核起始因子4E结合蛋白1（4E-BP1）的磷酸化，这是第二个mTOR依赖性事件，其效力与其对S6K1的作用相似。这种反应被雷帕霉素阻断，但不受100 nM渥曼青霉素的明显影响，这意味着mTOR和PI3K在S6K1激活中具有不同的作用。渥曼青霉素消除了卡巴胆碱刺激引起的PtdIns(3,4,5)P3升高，并大大降低了该脂质的基础水平。相比之下，表皮生长因子受体激酶抑制剂AG1478可阻止卡巴胆碱刺激的ErbB3反式激活、PI3K募集和1321N1细胞中的蛋白激酶B激活，其对S6K1激活的降低不超过30%。10 nM胰岛素可克服这种效应，胰岛素本身不会刺激S6K1，但可使细胞内PtdIns(3,4,5)P3浓度与单独使用卡巴胆碱时相当。这些观察结果区分了mTOR和PI3K在调节S6K1中的必要作用，但表明最小的PI3K活性足以允许其他激活因子（如增加的胞质Ca2+浓度，这对毒蕈碱受体介导的反应至关重要）刺激S6K1。此外，4E-BP1以及推测的mTOR可通过这些机制独立于PI3K激活进行调节。
