A549细胞中DBNLS232磷酸化调控丝状伪足形成及细胞迁移运动

DBNL^(S232)regulates formation of filopodia and promotes cell migration in A549 cells

目的分析脑发育调节蛋白样蛋白(Drebrin-like,DBNL)对肺腺癌细胞迁移运动的影响,及其特异位点磷酸化修饰在丝状伪足形成中的作用。方法基于shRNA表达载体建立稳定敲减DBNL表达的A549细胞,分析其对癌细胞迁移运动的影响;综合利用在线蛋白质组数据库、定点突变和间接免疫荧光分析A549细胞中过表达野生型DBNL及3个位点突变型(S146E、T165E、S232E和S232A)对微丝骨架形态、丝状伪足形成及细胞迁移运动的影响;通过免疫沉淀实验确定A549细胞中S232位点发生磷酸化修饰。结果肺癌细胞中稳定敲减DBNL表达显著抑制细胞迁移运动(P<0.001)。通过CPTAC蛋白组数据库分析发现,肺腺癌样本中相较于癌旁组织DBNL蛋白水平表达差异不明显(P>0.05);但3个丝氨酸/苏氨酸磷酸化位点S146、T165和S232修饰化水平明显上调(P<0.001)。野生型及模拟磷酸化突变体S146E、T165E、S232E过表达在A549细胞中,免疫荧光检测发现S232E富集于膜周凸起,且显著促进癌细胞丝状伪足形成(P<0.001);而表达磷酸化失活型S232A则诱导其弥散状分布在细胞质中,并抑制丝状伪足形成(P<0.01)。最后利用免疫共沉淀及通用型丝氨酸磷酸化抗体证明A549细胞中DBNL的S232位点被磷酸化修饰;并基于在线磷酸化序列激酶预测分析发现,p38和CDK5等激酶是S232位点的潜在修饰激酶。结论肺腺癌细胞A549中磷酸化DBNLS232调控丝状伪足形成并促进细胞迁移运动。

Objective To investigate the role of site-specific phosphorylation of adaptor protein Drebrin-like(DBNL)in regulating the formation of filopodia and the migration and movement of lung cancer A549 cells.Methods DBNL was stably knocked down by shRNA in A549 cells,and then its effects on migration and movement of lung cancer cells were analyzed.Online proteome database,site-directed mutagenesis and indirect immunofluorescence assay were used integratively to analyze the effect of wild type DBNL and 3 mutant types(S146E and T165E,S232E and S232A)on F-actin arrangement,formation of filopodia and migration and movement in A549 cells.Co-immunoprecipitation assay was applied to confirm phosphorylation of DBNL S232.Results DBNL knockdown significantly inhibited the migration and movement of A549 cells(P0.001).CPTAC database analysis found no significant difference in total protein of DBNL between the adjacent and primary tissues(P0.05),however,the phosphorylation of DBNL S146,T165 and S232 were predominantly increased in lung cancer tissue when compared to adjacent tissue(P0.001).In addition,indirect-immunofluorescence assay indicated that DBNL S232E which was enriched around the membrane that mimic phosphorylation improved the formation of filopodia and the movement of cells(P0.001),but not DBNL wild type and other mutations(T165E,S232E).Furthermore,unphosphorylated mutation DBNL S232A obviously suppressed the formation of filopodia(P0.01).Finally,prediction for phosphorylation on online found that p38 and CDK5 kinases could potentially phosphorylate DBNL S232.Conclusion Phosphorylation of DBNL S232 in lung cancer A549 cells promotes the formation of filopodia and enhances the movement of lung cancer cells.