PEAK1通过FAK-CTTN-Arp2/3信号调控细胞迁移的分子机制研究

PEAK1 is a non-receptor protein tyrosine kinase which localized at actin cytoskeleton and focal adhesion sites, playing a crucial role in the occurrence and metastasis of high metastatic tumors, but the molecular mechanism of PEAK1-promoted tumor metastasis remained unknown. Our previous study found that knockdown of PEAK1 leads to enlarged focal adhesion and dynamic defects of pseudopodia. We identified some interaction proteins of PEAK1 by using co-immunoprecipitation coupled with LC-MS/MS. In these candidate interaction proteins, CTTN that mediated actin polymerization was proved to interact with PEAK1. Non-labeling quantitative proteomics analysis showed that tyrosine phosphorylation of CTTN is down-regulated, but FAK Y397 is up-regulated in PEAK1 knockdown cells. Therefore, we speculate that CTTN-activated actin polymerization and FAK-mediated focal adhesion turnover in pseudopodia play an important role in the process of PEAK1-promoted cell migration. We will dissect the mechanism of PEKA1-motivated cell migration from the following aspects: 1) Reveal the interaction mechanism between PEAK1 and CTTN；2）The effect of PEAK1 on CTTN tyrosine phosphorylation and the function of PEAK1 regulated CTTN phosphomodification in CTTN-Arp2/3 complex mediated actin polymerization in pseudopodia; 3) The influence of PEKA1 on the cellular localization, interaction and tyrosine phosphorylation of focal adhesion related proteins, such as FAK, Paxillin, Talin. 4) The role of Src in PEAK1-regulated tyrosine phosphorylation of FAK containing focal adhesion. These results will provide a theoretical basis for the study of PEAK1 as a marker for the diagnosis and treatment of high metastatic tumor.

PEAK1是一个非受体酪氨酸蛋白激酶，在细胞Actin微丝、粘着斑和伪足等部位定位，可促进多种肿瘤细胞的迁移，其调控机制不明。我们发现：沉默PEAK1可阻碍粘着斑、Actin微丝和伪足的动态变化，抑制肿瘤细胞迁移；PEAK1与伪足蛋白CTTN相互作用；沉默PEAK1导致粘着斑蛋白聚集，下调CTTN、Paxillin、Vinculin和Src等蛋白的酪氨酸磷酸化，上调FAK Y397。据此，我们推测：PEAK1可能通过CTTN介导伪足Actin的聚合及FAK调节粘着斑的动态组装，调控肿瘤细胞的迁移。本项目将进一步分析PEAK1与CTTN的互作结构域，筛选PEAK1调控CTTN的酪氨酸磷酸化位点；探讨PEAK1对CTTN-Arp2/3介导Actin体外聚合的影响；研究Src在PEAK1调控粘着斑蛋白定位、相互作用和磷酸化中的作用。为PEAK1作为高转移肿瘤诊疗标志物及治疗靶点的研究提供理依据。

细胞运动参与了胚胎发育、体内平衡、肿瘤细胞转移、生物治疗等生理、病理过程，解析细胞运动的调控机制具有重要意义。本项目以细胞为模型，系统揭示了PEAK1调控细胞运动的分子机制，探索了PEAK1缺失小鼠脂肪酸代谢等生理问题。PEAK1敲降后，会导致细胞片状伪足减少、粘着斑变大和Actin变粗等；除PEAK1的激酶结构域外，N端的Actin结合结构域、中间调控结构域能促进细胞运动；Co-IP结合质谱分析，发现PEAK1与细胞骨架蛋白Cortactin、Myosin II具有相互作用；PEAK1的Proline-rich结构域和Cortactin的SH3结构域是互作必须结构域，且相互作用受FAK、Rock、Raf和PKC等蛋白激酶以及金属蛋白酶MMP的调控；PEAK1上调了 Cortactin Y421的磷酸，进而促进了Cortactin与Arp2/3复合体、Cofilin、N-WASP等细胞骨架蛋白的结合，影响F-Actin和细胞片状伪足的形成；伤口愈合、随机运动试验表明过表达或敲降Cortactin对PEAK1促进的细胞运动影响不大，但敲降PEAK1会抑制Cortactin过表达促进的细胞运动。为了进一步明确PEAK1、粘着斑的关系，我们进一步分析了粘着斑相关蛋白PEAK1与FAK、Myosin II蛋白之间的关系：PEAK1的酪氨酸磷酸化、粘着斑和Actin等部位的定位及促进细胞运动等均依赖FAK激酶活性。敲降PEAK1增加了粘着斑相关蛋白FAK、Talin、paxillin和p130Cas之间的相互作用，进而稳定了粘着斑。敲降PEAK1改变了应力纤维形成蛋白Myosin IIA/B在细胞骨架上的定位，破坏了由Myosin II蛋白建立的细胞极性；抑制Myosin II的ATPase活性可以消除敲降PEAK1导致的粘着斑变大和F-Actin增粗的表型；敲降Myosin II可抵抗敲降PEAK1抑制的细胞运动。由此，我们可以得出PEKA1调控细胞运动依赖Myosin IIB蛋白介导的细胞骨架重组。综上，PEAK1调控细胞运动是一个复杂的过程，但通过与细胞骨架结合蛋白的相互作用、及参与FAK介导的信号通路是其调控细胞运动的主要方式。

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