Bialkowska Katarzyna, Byzova Tatiana V, Plow Edward F
From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195.
From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195
J Biol Chem. 2015 Mar 6;290(10):6226-42. doi: 10.1074/jbc.M114.634436. Epub 2015 Jan 21.
The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin αIIbβ3, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), αIIbβ3 became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β3 integrin and kindlin-3 in focal adhesions, and enhanced β3 integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr(482) or Ser(484) was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476-485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin β3 activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs.
整合素对细胞反应的贡献取决于其激活状态，而激活是由蛋白质与整合素胞质尾部的结合所调控的。踝蛋白是整合素胞质尾部的结合伴侣，对整合素的最佳激活至关重要，而踝蛋白-3在造血细胞中发挥这一作用。在此，我们使用表达整合素αIIbβ3的人血小板和人红白血病（HEL）细胞，来研究踝蛋白-3的磷酸化是否调节整合素激活。当用血小板生成素或佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）刺激HEL细胞时，αIIbβ3被激活，这可通过激活特异性单克隆抗体和可溶性纤维蛋白原的结合、在纤维蛋白原上的黏附与铺展、β3整合素和踝蛋白-3在黏着斑中的共定位以及免疫沉淀中β3整合素-踝蛋白-3结合增强来证明。敲低踝蛋白-3会损害在纤维蛋白原上的黏附与铺展。用激动剂刺激HEL细胞可显著增加通过质谱测序检测到的踝蛋白-3磷酸化。苏氨酸（Thr）482或丝氨酸（Ser）484被鉴定为磷酸化位点，该位点位于踝蛋白-1或踝蛋白-2中不保守的序列中。当用凝血酶刺激血小板时，踝蛋白-3中的这些相同残基也会被磷酸化。当在HEL细胞中表达时，与野生型踝蛋白-3相比，T482A/S484A踝蛋白-3降低了可溶性配体结合以及在纤维蛋白原上的细胞铺展。将包含踝蛋白-3第476 - 485位残基的膜渗透性肽导入HEL细胞和血小板中；与乱序对照肽相比，两种细胞类型的黏附与铺展均受到抑制。这些数据确定了踝蛋白-3磷酸化在整合素β3激活中的作用，并为踝蛋白-3与其他两种踝蛋白旁系同源物之间的功能差异提供了基础。
