背景:局灶性粘连(FAs)含有整合素,多蛋白结构,将细胞内肌动蛋白连接到细胞外基质,并触发控制细胞增殖的多个信号通路,分化,生存和运动。微管(MT)通过与皮质微管稳定复合物(CMSC)的成分相互作用而稳定在FA附近。CMSC内的KANK(KN基序和锚蛋白重复结构域)家族蛋白,KANK1或KANK2在FAs内结合talin,从而介导肌动蛋白-MT串扰。我们先前在MDA-MB-435S细胞中鉴定,优先使用整合素αVβ5进行粘附,KANK2作为实现肌动蛋白-MT串扰的关键分子。KANK2敲除还导致对MT毒物的敏感性增加,紫杉醇(PTX)和长春新碱和减少迁移。这里,我们的目的是分析KANK1是否具有相似的作用,并区分哪种talin亚型与KANK2结合.
方法:细胞模型由人黑色素瘤细胞系MDA-MB-435S和整合素αV(3αV)表达降低的稳定转染克隆组成。对于talin1、talin2、KANK1或KANK2的瞬时敲低,我们使用基因特异性siRNA转染。使用先前标准化的方案,我们分离了整联蛋白粘附复合物。SDS-PAGE和Western印迹用于蛋白质表达分析。使用共聚焦显微镜进行免疫荧光分析和活细胞成像。用Transwell细胞培养插入物分析细胞迁移。使用GraphPad软件的统计分析包括任一单向方差分析(ANOVA),不成对学生t检验或双向方差分析。
结果:我们表明KANK1不是与整合素αVβ5FAs相关的CMSC的一部分,其敲除不影响MT生长的速度或细胞对PTX的敏感性。talin2敲低模仿KANK2敲低,即导致肌动蛋白-MT串扰的扰动,这表明MT生长的速度增加,对PTX的敏感性增加,迁移也减少。
结论:我们得出结论,KANK2在功能上与talin2相互作用,并且对PTX敏感性增加的机制涉及微管动力学的变化。这些数据阐明了talin2和KANK2同种型的细胞类型特异性作用,因此我们认为talin2和KANK2是改善癌症治疗的潜在治疗靶标。
BACKGROUND: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVβ5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2.
METHODS: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student\'s t-test or two-way ANOVA analysis.
RESULTS: We show that KANK1 is not a part of the CMSC associated with integrin αVβ5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration.
CONCLUSIONS: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.