Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca(2+)-transients in human platelets. Role of a cdc2-related protein kinase.
Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca(2+)-transients in human platelets. Role of a cdc2-related protein kinase.
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1 型和 2A 型蛋白磷酸酶抑制剂可减弱人血小板中的磷脂酰肌醇代谢和 Ca(2) 瞬态。
DOI：
10.1021/bi00143a027
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Lerea,KM
中科院分区：
文献类型：
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作者：
Lerea,KM
细胞生物学和解剖学，纽约医学院基础科学大楼，瓦尔哈拉，纽约10595摘要：加入冈田酸或钙素A可使人血小板对凝血酶脱敏。本研究的目的之一是确定导致分泌反应的哪个步骤可能受到这些蛋白磷酸酶抑制剂的影响。以剂量依赖性的方式，冈田酸或钙调素a抑制磷脂酰肌醇代谢和Ca2+瞬态。在所有情况下，花青素A的效力大约是冈田酸的10倍，并且在1 pM的浓度下效果最大。虽然凝血酶诱导的[Ca2+] j升高减少，但仍观察到肌球蛋白轻链（MLC）磷酸化状态的增加。然而，在将凝血酶添加到含有二甲基bapta的calyculin a处理过的血小板后，这种磷酸化的变化减弱了。这些数据表明，花青素A和冈田酸降低激动剂诱导的Ca2+瞬态，这反过来又阻止反应，如分泌反应。Calyculin A/冈田酸诱导的磷酸化事件在装载bapta的血小板中没有减少，这表明这些磷酸化是Ca2+不敏感的。因此，本研究的第二个目标是确定负责calyculin a诱导磷酸化的蛋白激酶。在血小板裂解液系统中，calyculin a导致p50中[32P]磷酸盐的掺入增加。这种磷酸化事件与在完整血小板中观察到的相同，并且没有被cAMP， cGMP， Ca2+或Ca2+/磷脂/二酰基甘油混合物模仿。将裂解物与pl3sul - sepharose孵育后，去除激酶活性。这表明一个pl3sul敏感的蛋白激酶，例如，一个细胞周期依赖的蛋白激酶，负责calyculin a敏感的磷酸化事件。为了支持这一观点，我们在pl3sucl-Sepharose沉淀物中检测到cdk2，使用的抗体是为了保护激酶的区域而产生的。A.激动剂和拮抗剂改变血小板蛋白的磷酸化状态（Lyons et al., 1975; Haslam et al., 1979）。因此，许多实验室对蛋白磷酸化与血小板反应调节之间的相关性进行了大量研究[见Siess(1989)]。然而，蛋白质磷酸化在多大程度上调节血小板反应仍然需要澄清。研究表明，有限数量蛋白的磷酸化状态反映了血小板的激活状态(Kawamoto et al.；
Department of Cell Biology and Anatomy, Basic Sciences Building, New York Medical College, Valhalla, New York 10595 Received February 12, 1992; Revised Manuscript Received April 17, 1992 abstract: The addition of either okadaic acid or calyculin A desensitizes human platelets to thrombin. One objective of this study was to determine which step (s) leading to secretion reactions may be affected by these protein phosphatase inhibitors. In a dose-dependent manner, okadaic acid or calyculin A inhibits phosphatidylinositol metabolism and Ca2+-transients. In all cases, calyculin A was approximately 10-fold more potent than okadaic acid, and it had maximal effects at a concentration of 1 pM. Although throm-bin-induced rises in [Ca2+] j were diminished, an increase in the phosphorylationstate of myosin light chains (MLC) was still observed. Changes in this phosphorylation were diminished, however, following the addition of thrombin to calyculin A-treated platelets that were loaded with dimethyl-BAPTA. These data demonstrate that calyculin A and okadaic acid lower agonist-induced Ca2+-transients, which in turn prevents responses such as secretion reactions. Calyculin A/okadaic acid-induced phosphorylation events were not diminished in BAPTA-loaded platelets, suggesting that these phosphorylations are Ca2+-insensitive. Thus, a second objective of this study was to identify the protein kinase (s) that was (were) responsible for the calyculin A-induced phosphorylations. In a platelet lysate system, calyculin A caused an increase in the incorporation of [32P] phosphate into p50. This phosphorylation event was identical to that observed in the intact platelet and was not mimicked by cAMP, cGMP, Ca2+, or a Ca2+/phospholipid/diacylglycerol mixture. Kinase activity was removed after the lysate was incubated with pl3sucl-Sepharose. This suggests that a pl3sucl-sensitive protein kinase, eg, a cell cycle-dependent protein kinase, is responsible for the calyculin A-sensitive phosphorylation events. To support this notion, cdk2 was detected in pl3sucl-Sepharose precipitates using antibodies that were generated to conserve regions of the kinase.. A. gonists and antagonists alter the phosphorylation state of platelet proteins (Lyons et al., 1975; Haslam et al., 1979). Consequently, the correlation between protein phosphorylations and regulation of platelet responses has been investigated vigorously by many laboratories [reviewed in Siess (1989)]. However, the extent to which protein phosphorylation regulates platelet responses still needs to be clarified. Studies have shown that the phosphorylationstatus of a limited number of proteins reflects the activation state of platelets (Kawamoto et al.,