Identification of Novel Gene Targets and Functions of p21-Activated Kinase 1 during DNA Damage by Gene Expression Profiling

P21-activated kinase 1 (PAK1), a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR), and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new aspects of PAK1 biology.P21激活激酶1（P21-activated kinase 1，PAK1）作为一种丝氨酸/苏氨酸蛋白激酶（serine/threonine protein kinase），可通过磷酸化下游底物调控诸多细胞生物学过程。除在细胞质中发挥功能外，PAK1还可通过核定位以及与染色质（chromatin）的结合影响基因转录。目前已有研究证实，电离辐射（ionizing radiation，IR）可快速激活PAK1的激酶活性并促进其核转位，而PAK1激活是DNA损伤应答（DNA damage response）的组成部分之一。鉴于PAK1在细胞存活中的作用、其与染色质的结合特性，以及当前发现的电离辐射刺激效应，我们提出假说：PAK1可能参与调控那些在DNA损伤应答中发挥关键作用的细胞生物学过程相关基因。本研究旨在筛选出在电离辐射应答中、且可能参与DNA损伤应答的新型PAK1靶基因。本研究借助微阵列（microarray）技术，检测了电离辐射对有无Pak1表达的小鼠胚胎成纤维细胞基因表达谱的影响。采用Gene Spring GX 10.0.2软件筛选得到差异表达转录本。分别通过京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）、Ingenuity Pathway、基因本体（Gene Ontology）以及PANTHER数据库，完成通路、网络、功能富集分析及基因家族分类。通过实时定量聚合酶链式反应（RT-qPCR）验证了部分PAK1靶基因的表达水平。本研究首次完成了PAK1的全基因组分析，并筛选出了可能参与DNA损伤应答的PAK1靶基因。基因本体分析显示，电离辐射刺激后的细胞中，差异表达基因主要参与细胞周期阻滞与细胞死亡过程。通路富集分析结果表明，p53通路是受电离辐射应答及PAK1靶基因调控最为显著的通路。转录因子基因家族呈现显著富集，同时还筛选出了参与DNA复制、修复及细胞信号转导的基因调控网络。综上，本研究筛选得到了依赖于PAK1的新型电离辐射应答基因，为揭示PAK1的生物学功能提供了新的视角。