Buschbeck M, Ghomashchi F, Gelb M H, Watson S P, Börsch-Haubold A G
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
Biochem J. 1999 Dec 1;344 Pt 2(Pt 2):359-66.
Stress stimuli such as free radicals, high osmolarity or arsenite activate stress-activated protein kinases (SAPKs) in a wide variety of cells. In the present study, we have investigated the ability of several stress stimuli to activate SAPKs in platelets and to induce phosphorylation of their substrates. Treatment of human platelets with H(2)O(2) stimulated SAPK2a and its downstream target mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). Kinase activity reached a maximum after 2-5 min and declined towards basal levels after 15 min. Arsenite caused a steady increase of MAPKAP-K2 activity up to 15 min. The level of maximal kinase activation by H(2)O(2) and arsenite was comparable with the effect caused by the physiological platelet stimulus thrombin. A high osmolarity solution of sorbitol induced comparatively small activation of SAPK2a and MAPKAP-K2. The 42-kDa extracellular signal-regulated kinase (ERK) 2 was not activated by H(2)O(2), sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H(2)O(2) and sorbitol enhanced the release of arachidonic acid induced by the calcium ionophore A23187. This effect was reversed by the inhibitor of SAPK2a, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) imidazole (SB 203580), but not by the inhibitor of the ERK2-activating pathway, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one (PD 98059). Both H(2)O(2) and sorbitol increased phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA(2) by H(2)O(2) occurred on Ser-505, a reaction that is known to increase the intrinsic lipase activity of the enzyme. Our results demonstrate that activation of SAPKs by stress stimuli primes cPLA(2) activation through phosphorylation. In vivo, this mechanism would lead to the sensitization of platelet activation and may be an important risk factor in thrombotic disease.
诸如自由基、高渗透压或亚砷酸盐等应激刺激可在多种细胞中激活应激激活蛋白激酶（SAPKs）。在本研究中，我们调查了几种应激刺激激活血小板中SAPKs及其诱导底物磷酸化的能力。用H₂O₂处理人血小板可刺激SAPK2a及其下游靶点丝裂原活化蛋白激酶激活的蛋白激酶-2（MAPKAP-K2）。激酶活性在2 - 5分钟后达到最大值，并在15分钟后降至基础水平。亚砷酸盐导致MAPKAP-K2活性持续增加直至15分钟。H₂O₂和亚砷酸盐引起的最大激酶激活水平与生理性血小板刺激物凝血酶所产生的效果相当。高渗山梨醇溶液诱导的SAPK2a和MAPKAP-K2激活相对较小。42 kDa的细胞外信号调节激酶（ERK）2未被H₂O₂、山梨醇或亚砷酸盐激活。这些刺激单独均未引发显著的花生四烯酸释放。然而，H₂O₂和山梨醇增强了钙离子载体A23187诱导的花生四烯酸释放。SAPK2a抑制剂4 -（4 - 氟苯基）- 2 -（4 - 甲亚磺酰基苯基）- 5 -（4 - 吡啶基）咪唑（SB 203580）可逆转此效应，但ERK2激活途径抑制剂2 -（2 - 氨基 - 3 - 甲氧基苯基）- 氧杂萘 - 4 - 酮（PD 98059）则不能。H₂O₂和山梨醇均增加了胞质磷脂酶A₂（cPLA₂）的磷酸化及其内在活性；这两种反应均被SB 203580阻断。H₂O₂诱导的cPLA₂磷酸化发生在Ser - 505位点，已知该反应会增加该酶的内在脂肪酶活性。我们的结果表明，应激刺激激活SAPKs通过磷酸化引发cPLA₂激活。在体内，这种机制将导致血小板激活的敏化，可能是血栓性疾病的一个重要危险因素。
