Synergistic activation of tyrosine phosphorylation by o-vanadate plus calcium ionophore A23187 or aromatic 1,2-diols.
Synergistic activation of tyrosine phosphorylation by o-vanadate plus calcium ionophore A23187 or aromatic 1,2-diols.
复制标题
邻钒酸盐加钙离子载体 A23187 或芳香族 1,2-二醇对酪氨酸磷酸化的协同激活。
DOI：
10.1021/bi00172a031
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Earp,HS
中科院分区：
文献类型：
--
作者：
Huckle,WR;Earp,HS
1993年11月8日收到的修订手稿·摘要：我们之前已经证明，用Ca 2+离子载体A23187处理WB大鼠肝上皮细胞会引起蛋白质酪氨酸磷酸化的快速增加，从而忠实地再现了血管紧张素II所观察到的Ca 2+依赖性反应。在酪氨酸磷酸酶抑制剂o-钒酸盐（2.0-200 μ M）的存在下，对A23187的酪氨酸磷酸化反应的幅度增加> 10倍。A23187和钒酸盐的这种协同作用明显不同于血管紧张素II和钒酸盐的联合作用，后者仅仅是相加作用。无论是细胞外或细胞内的钙螯合废除协同响应离子载体和钒酸盐，表明其钙依赖性。通过对WB细胞经致癌剂处理后转化的GN 4细胞系的研究，明确表明血管紧张素II和A23187/钒酸盐反应中涉及不同的途径。GN 4细胞对血管紧张素非依赖性酪氨酸激酶激活的反应性是WB细胞的2-3倍，但它们完全缺乏对A23187/钒酸盐的协同酪氨酸磷酸化反应。为了测试花生四烯酸代谢产物在A23187/钒酸盐反应中的作用，用吲哚美辛或去甲二氢愈创木酸（NDGA）预处理细胞。这两种化合物都没有抑制作用，但令人惊讶的是，NDGA加钒酸盐密切模仿WB细胞中的A23187/钒酸盐反应，并且像A23187/钒酸盐一样，在GN 4细胞中无效。NDGA含有儿茶酚核（即芳香族1，2-二醇），其中类似于类黄酮抗氧化剂槲皮素，另一种发现与钒酸盐协同增加酪氨酸磷酸化的化合物。在我们的研究中，含有1，2-二醇的类黄酮（槲皮素、非瑟酮）与钒酸盐协同升高P-Tyr（在WB中而不是在GN 4细胞中），而缺乏1，2-二醇的类黄酮（芹菜素、桑色素）是无活性的。用谷胱甘肽前体7V-乙酰半胱氨酸过夜预处理抑制了对A23187/钒酸盐和NDGA/钒酸盐的反应。我们推测，儿茶酚与钒酸盐的组合，单独或作为螯合复合物，可能会增强酪氨酸磷酸酶抑制钒酸盐或可能会延迟代谢失活的钒酸盐[例如，还原为V（IV）的谷胱甘肽]。同样，A23187引起的钙持续升高可能改变细胞内的氧化还原状态，可能是通过细胞内谷胱甘肽的耗竭。由于酪氨酸磷酸酶是精致敏感的巯基氧化和潜在的监管氧化状态的o-钒酸盐，这些结果加强的概念，即局部调节细胞内的氧化还原状态可能是一个重要的决定因素酪氨酸磷酸酶activities.Phosphorylation的酪氨酸残基上的蛋白质是一个经常与控制细胞增殖的调节机制。酪氨酸磷酸化蛋白质的稳态水平由酪氨酸激酶和磷酸酶（PTPases）的相反作用决定，PTPases是一种在非常大且多样的家族中发现的酶。例如，酪氨酸激酶和PTPases
Revised Manuscript Received November 8, 1993• abstract: We have shown previously that treatment of WB rat liver epithelial cells with the Ca2+ ionophore A23187 provokes a rapid increase in protein-tyrosine phosphorylation that faithfully reproduces the Ca2+-dependent response seen with angiotensin II. In the presence of the tyrosine phosphatase inhibitor o-vanadate (2.0-200 juM), the tyrosine phosphorylation response to A23187 was increased> 10-fold in magnitude. This synergistic effect of A23187 and vanadate is clearly distinct from the combined effect of angiotensin II and vanadate, which was merely additive. Chelation of either extracellular or intracellular Ca2+ abolished the synergistic response to ionophore and vanadate, indicating its Ca2+ dependence. That divergent pathways were involved in the angiotensin II and the A23187/vanadate responses was shown definitely by studies of GN4 cells, a transformed line derived from WB cells by carcinogen treatment. GN4 cells are 2-3-fold more responsive thanWB cells to angiotensin Independent tyrosine kinase activation, yet they completely lacked the synergistic tyrosine phosphorylation response to A23187/vanadate. To test the role of arachidonic acid metabolites in the A23187/vanadate response, cells were pretreated with eitherindomethacin or nordihydroguaiaretic acid (NDGA). Neither compound was inhibitory, but surprisingly, NDGA plus vanadate closely mimicked the A23187/vanadate response inWB cells and, like A23187/vanadate, was ineffective in GN4 cells. NDGA contains catechol nuclei (ie, aromatic 1, 2-diols) and therein resembles the flavonoidanti-oxidant quercetin, another compound found to increase tyrosine phosphorylation synergistically with vanadate. In our studies, flavonoids that contain 1, 2-diols (quercetin, fisetin) were synergistic with vanadate in elevating P-Tyr (in WB but not GN4 cells) while those lacking 1, 2-diols (apigenin, morin) were inactive. Overnight pretreatment with a glutathione precursor, 7V-acetylcysteine, inhibited the responses to A23187/vanadate and NDGA/vanadate. We postulate that combinations of catechols with vanadate, separately or as chelation complexes, may potentiate tyrosinephosphatase inhibition by vanadate or may delay the metabolic inactivation of vanadate [eg, reduction to V (IV) by glutathione]. Similarly, the sustained elevation of calcium caused by A23187 maychange intracellular redox status, perhaps by the depletion of intracellular glutathione. Since tyrosine phosphtases are exquisitely sensitive to sulfhydryl oxidation and potentially to the regulated oxidative state of o-vanadate, these results reinforce the concept that local regulation of intracellular redox state may be an important determinant of tyrosine phosphatase activities.Phosphorylation of proteins on tyrosine residues is a regulatory mechanism frequently associated with the control of cell proliferation. Steady-state levels of tyrosine-phosphorylated proteins are determined by the opposing actions of tyrosine kinases and phosphatases (PTPases), 1 enzymes that have been found to occur in remarkably large and diverse families. For example, both tyrosine kinases and PTPases