Maślikowski Bart M, Wang Lizhen, Wu Ying, Fielding Ben, Bédard Pierre-André
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Department of Biology, McMaster University, Hamilton, Ontario, Canada
J Virol. 2016 Dec 16;91(1). doi: 10.1128/JVI.01925-16. Print 2017 Jan 1.
The increase in AP-1 activity is a hallmark of cell transformation by tyrosine kinases. Previously, we reported that blocking AP-1 using the c-Jun dominant negative mutant TAM67 induced senescence, adipogenesis, or apoptosis in v-Src-transformed chicken embryo fibroblasts (CEFs) whereas inhibition of JunD by short hairpin RNA (shRNA) specifically induced apoptosis. To investigate the role of AP-1 in Src-mediated transformation, we undertook a gene profiling study to characterize the transcriptomes of v-Src-transformed CEFs expressing either TAM67 or the JunD shRNA. Our study revealed a cluster of 18 probe sets upregulated exclusively in response to AP-1/JunD impairment and v-Src transformation. Four of these probe sets correspond to genes involved in the interferon pathway. One gene in particular, death-associated protein kinase 1 (DAPK1), is a C/EBPβ-regulated mediator of apoptosis in gamma interferon (IFN-γ)-induced cell death. Here, we show that inhibition of DAPK1 abrogates cell death in v-Src-transformed cells expressing the JunD shRNA. Chromatin immunoprecipitation data indicated that C/EBPβ was recruited to the DAPK1 promoter while the expression of a dominant negative mutant of C/EBPβ abrogated the induction of DAPK1 in response to the inhibition of AP-1. In contrast, as determined by chromatin immunoprecipitation (ChIP) assays, JunD was not detected on the DAPK1 promoter under any conditions, suggesting that JunD promotes survival by indirectly antagonizing the expression of DAPK1 in v-Src transformed cells.
Transformation by the v-Src oncoprotein causes extensive changes in gene expression in primary cells such as chicken embryo fibroblasts. These changes, determining the properties of transformed cells, are controlled in part at the transcriptional level. Much attention has been devoted to transcription factors such as AP-1 and NF-κB and the control of genes associated with a more aggressive phenotype. In this report, we describe a novel mechanism of action determined by the JunD component of AP-1, a factor enhancing cell survival in v-Src-transformed cells. We show that the loss of JunD results in the aberrant activation of a genetic program leading to cell death. This program requires the activation of the tumor suppressor death-associated protein kinase 1 (DAPK1). Since DAPK1 is phosphorylated and inhibited by v-Src, these results highlight the importance of this kinase and the multiple mechanisms controlled by v-Src to antagonize the tumor suppressor function of DAPK1.
AP-1活性增加是酪氨酸激酶介导细胞转化的一个标志。此前，我们报道过，使用c-Jun显性负性突变体TAM67阻断AP-1可诱导v-Src转化的鸡胚成纤维细胞（CEF）发生衰老、脂肪生成或凋亡，而短发夹RNA（shRNA）抑制JunD则特异性诱导凋亡。为了研究AP-1在Src介导的转化中的作用，我们进行了一项基因谱分析研究，以表征表达TAM67或JunD shRNA的v-Src转化CEF的转录组。我们的研究揭示了一组18个探针集，它们仅在对AP-1/JunD损伤和v-Src转化的反应中上调。其中四个探针集对应于参与干扰素途径的基因。特别是一个基因，死亡相关蛋白激酶1（DAPK1），是γ干扰素（IFN-γ）诱导的细胞死亡中C/EBPβ调节的凋亡介质。在这里，我们表明抑制DAPK1可消除表达JunD shRNA的v-Src转化细胞中的细胞死亡。染色质免疫沉淀数据表明C/EBPβ被募集到DAPK-1启动子，而C/EBPβ显性负性突变体的表达消除了对AP-1抑制的反应中DAPK1的诱导。相反，通过染色质免疫沉淀（ChIP）分析确定，在任何条件下DAPK1启动子上均未检测到JunD，这表明JunD通过间接拮抗v-Src转化细胞中DAPK1的表达来促进细胞存活。
v-Src癌蛋白介导的转化会导致原代细胞如鸡胚成纤维细胞的基因表达发生广泛变化。这些变化决定了转化细胞的特性，部分在转录水平受到控制。人们对转录因子如AP-1和NF-κB以及与更具侵袭性表型相关的基因控制给予了很多关注。在本报告中，我们描述了由AP-1的JunD成分决定的一种新作用机制，AP-1是一种增强v-Src转化细胞中细胞存活的因子。我们表明JunD的缺失导致导致细胞死亡的遗传程序异常激活。该程序需要肿瘤抑制因子死亡相关蛋白激酶1（DAPK1）的激活。由于DAPK1被v-Src磷酸化并抑制，这些结果突出了该激酶的重要性以及v-Src控制的多种机制对拮抗DAPK1肿瘤抑制功能的作用。
