Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides
Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides
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DOI：
10.1091/mbc.10.8.2669
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发表时间：
1999-08-01
影响因子：
3.3
通讯作者：
Furthmayr, H
中科院分区：
文献类型：
--
作者：
Nakamura, F;Huang, LQ;Furthmayr, H
用凝血酶激活人血小板会暂时增加 moesin (558) 苏氨酸的磷酸化，如磷酸化状态特异性抗体所测定。这种特定的修饰被激酶抑制剂十字孢菌素完全抑制，并被磷酸酶抑制剂花萼蛋白 A 最大限度地促进，从而可以将两种形式的 moesin 纯化至均质。使用标记有 [P-32]ATP 或 I-125 的 F-肌动蛋白探针进行的印迹重叠测定表明，只有磷酸化的 moesin 与总血小板裂解物、moesin 抗体免疫沉淀物中的 F-肌动蛋白相互作用，并且在纯化时，在没有去污剂的情况下，两种形式的分离蛋白都会聚集。磷酸化、纯化的模蛋白与 α- 或 β/γ- 肌动蛋白丝在阳离子洗涤剂中共沉淀，但不在阴离子、非离子或两性洗涤剂中。相互作用亲和力很高（K-d，类似于 1.5 nM），最大 moesin:actin 化学计量为 1:1。在用阳离子洗涤剂而非非离子洗涤剂提取的血小板中也观察到这种相互作用。在 0.1% Triton X-100 中，F-肌动蛋白仅在存在多磷脂酰肌醇的情况下才与磷酸化 moesin 相互作用。因此，多磷脂酰肌醇和磷酸化都可以在体外激活moesin的高亲和力F-肌动蛋白结合位点。两种机制的双重调节对于肌动蛋白细胞骨架和质膜之间由莫斯蛋白介导的连接的正确细胞控制可能很重要。
Activation of human platelets with thrombin transiently increases phosphorylation at (558)threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [P-32]ATP or I-125 show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified, in the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with alpha- or beta/gamma-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (K-d, similar to 1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. in 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin's high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.