Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute Cleveland Clinic, Cleveland, OH, USA.
Department of Molecular Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.
Life Sci Alliance. 2020 Feb 5;3(3). doi: 10.26508/lsa.201900594. Print 2019 Mar.
Studies of isolated cells, mice, and humans have demonstrated the vital role of the FERM domain protein kindlin-3 in integrin activation in certain hematopoietic and non-hematopoietic cells, consequent to binding to integrin β-subunits. To explore regulatory mechanisms, we developed a monoclonal antibody that selectively recognizes the phosphorylated form of Ser (pS) in kindlin-3. Activation of platelets, HEL megakaryocytic-like cells and BT549 breast cancer cells led to enhanced expression of pS as assessed by immunofluorescence or Western blotting. In platelets, pS rose rapidly and transiently upon stimulation. When a mutant form of kindlin-3, TS/AA kindlin-3, was transduced into mouse megakaryocytes, it failed to support activation of integrin αβ, whereas wild-type kindlin-3 did. In MDA-MB231 breast cancer cells, expression of TS/AA kindlin-3 suppressed cell spreading, migration, invasion, and VEGF production. Wild-type kindlin-3 expressing cells markedly increased tumor growth in vivo, whereas TS/AA kindlin-3 significantly blunted tumor progression. Thus, our data establish that a unique phosphorylation event in kindlin-3 regulates its cellular functions.
已有研究表明，在某些造血细胞和非造血细胞中，衔接蛋白 β 亚单位与 FERM 结构域蛋白——连接蛋白-3（kindlin-3）结合后，连接蛋白-3 可作为整合素激活的关键因子。为了研究其调控机制，我们开发了一种单克隆抗体，该抗体可特异性识别连接蛋白-3 中丝氨酸（Ser）磷酸化形式（pS）。免疫荧光或 Western blot 检测到，血小板、HEL 巨核细胞样细胞和 BT549 乳腺癌细胞的激活会导致 pS 表达增强。在血小板中，刺激后 pS 迅速短暂地升高。当向小鼠巨核细胞转导连接蛋白-3 的 TS/AA 突变形式时，其无法支持整合素 αβ 的激活，而野生型连接蛋白-3 则可以。在 MDA-MB231 乳腺癌细胞中，TS/AA 连接蛋白-3 的表达抑制了细胞铺展、迁移、侵袭和 VEGF 的产生。表达野生型连接蛋白-3 的细胞在体内显著增加了肿瘤的生长，而 TS/AA 连接蛋白-3 则显著抑制了肿瘤的进展。因此，我们的数据证实了连接蛋白-3 中的一个独特磷酸化事件可调节其细胞功能。
