AKT serine/threonine kinase 2-mediated phosphorylation of fascin threonine 403 regulates esophageal cancer progression
AKT serine/threonine kinase 2-mediated phosphorylation of fascin threonine 403 regulates esophageal cancer progression
复制标题
AKT 丝氨酸/苏氨酸激酶 2 介导的肌成束蛋白苏氨酸 403 磷酸化调节食管癌进展
DOI：
10.1016/j.biocel.2022.106188
复制
发表时间：
2022-03-01
影响因子：
4
通讯作者：
Li, En-Min
中科院分区：
文献类型：
--
作者：
Zhang, Zhi-Da;Wen, Bing;Li, En-Min
Fascin是丝状伪足中主要的肌动蛋白捆绑蛋白，并且在转移性肿瘤细胞中高度表达。Fascin的过度表达与不良的临床预后和转移进展相关。Fascin的翻译后修饰，如磷酸化，可以通过调节Fascin的肌动蛋白集束活性来影响肿瘤细胞的增殖和侵袭。然而，Fascin及其相应激酶的磷酸化位点需要进一步探索。在当前的研究中，我们鉴定了由AKT丝氨酸/苏氨酸激酶2（AKT 2）介导的Fascin Threatin 403（Fascin-T403）的新型磷酸化，其使用来自食管癌组织的质谱数据进行了研究（iProX数据库：IPX 0002501000）。分子动力学模拟结果显示，Fascin-T403 D磷酸化蛋白具有明显的氨基酸残基空间结构和相关性，与野生型Fascin-WT蛋白不同。低速离心实验结果表明Fascin-T403 D影响肌动蛋白的交联。为了研究Fascin-T403 D是否影响食管癌细胞的功能，在Fascin敲除或siRNA细胞系中拯救Fascin-WT或FascinT 403 D。我们观察到Fascin-T403 D可以抑制食管癌细胞的生物学行为，包括丝状伪足形成，细胞增殖和迁移。进行免疫共沉淀（Co-IP）和Duolink原位邻近连接测定（PLA）以测量Fascin和AKT 2之间的相互作用。使用体外和体内激酶测定，我们证实了AKT 2，而不是AKT 1或AKT 3，是Fascin Threatin 403的上游激酶。总之，AKT 2催化的肌成束蛋白Thr 403磷酸化抑制食管癌细胞行为、肌动蛋白捆绑活性和丝状伪足形成。
Fascin is the main actin-bundling protein in filopodia and is highly expressed in metastatic tumor cells. The overexpression of Fascin has been associated with poor clinical prognosis and metastatic progression. Post translational modifications of Fascin, such as phosphorylation, can affect the proliferation and invasion of tumor cells by regulating the actin-bundling activity of Fascin. However, the phosphorylation sites of Fascin and their corresponding kinases require further exploration. In the current study, we identified novel phosphorylation of Fascin Threonine 403 (Fascin-T403) mediated by AKT serine/threonine kinase 2 (AKT2), which was studied using mass spectrometry data from esophageal cancer tissues (iProX database: IPX0002501000). A molecular dynamics simulation revealed that Fascin-Threonine 403 phosphorylation (Fascin-T403D) had a distinct spatial structure and correlation of amino acid residues, which was different from that of the wild type (Fascin-WT). Low-speed centrifugation assay results showed that Fascin-T403D affected actin cross-linking. To investigate whether Fascin-T403D affected the function of esophageal cancer cells, either Fascin-WT or FascinT403D were rescued in Fascin-knockout or siRNA cell lines. We observed that Fascin-T403D could suppress the biological behavior of esophageal cancer cells, including filopodia formation, cell proliferation, and migration. Co-immunoprecipitation (Co-IP) and Duolink in situ proximity ligation assay (PLA) were performed to measure the interaction between Fascin and AKT2. Using in vitro and in vivo kinase assays, we confirmed that AKT2, but not AKT1 or AKT3, is an upstream kinase of Fascin Threonine 403. Taken together, the AKT2-catalyzed phosphorylation of Fascin Threonine 403 suppressed esophageal cancer cell behavior, actin-bundling activity, and filopodia formation.