Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.
Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.
复制标题
通过胞外蛋白激酶/磷酸酶系统对人血小板表面蛋白进行磷酸化和去磷酸化。
DOI：
10.1016/0167-4889(91)90165-t
复制
发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Ehrlich,YH
中科院分区：
文献类型：
--
作者：
Naik,UP;Kornecki,E;Ehrlich,YH
我们在人血小板膜表面发现了一种新的外蛋白激酶活性和一种新的外蛋白磷酸酶活性。洗净的完整血小板，在Tyrode缓冲液中与[γ-32P]ATP孵育，显示膜表面蛋白磷酸化，在5-15% SDS聚丙烯酰胺梯度凝胶上以42 kDa的表观分子质量迁移。42 kDa蛋白在15% SDS凝胶上进一步分离为39 kDa和42 kDa两种蛋白。在该凝胶体系中，发现39 kDa蛋白被快速磷酸化和去磷酸化，而42 kDa蛋白被磷酸化和去磷酸化的速度要慢得多。NaF抑制了这些蛋白的去磷酸化，表明参与了外蛋白磷酸酶。负责这两种蛋白磷酸化的血小板膜外蛋白激酶被确定为丝氨酸激酶，并显示出对二价阳离子Mg2+或Mn2+离子的依赖性。Ca2+离子增强了Mg2+依赖性外蛋白激酶的活性。外蛋白激酶迅速磷酸化组蛋白和酪蛋白外源性添加到完整血小板的细胞外培养基中。α-凝血酶激活血小板后，内源性蛋白底物对外源[γ-32P]ATP [32P]磷酸盐的掺入减少了90%，提示外蛋白激酶系统在血小板功能调节中的作用。结果表明，蛋白激酶和蛋白磷酸酶活性存在于人血小板的膜表面。这些活性能够快速磷酸化和去磷酸化特异性表面血小板膜蛋白，这可能在血小板激活和分泌的早期事件中起重要作用。
We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [γ-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5–15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+or Mn2+ions. Ca2+ions potentiated the Mg2+-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by α-thrombin, the incorporation of [32P]phosphate from exogenously added [γ-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.