title: JunD/AP-1 Antagonizes the Induction of DAPK1 To Promote the Survival of v-Src-Transformed Cells.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors: Department of Biology, McMaster University, Hamilton, Ontario, Canada.
authors: Bart M
authors: BM
authors: Maślikowski
authors: Department of Biology, McMaster University, Hamilton, Ontario, Canada.
authors: Lizhen
authors: L
authors: Wang
authors: Department of Biology, McMaster University, Hamilton, Ontario, Canada.
authors: Ying
authors: Y
authors: Wu
authors: Department of Biology, McMaster University, Hamilton, Ontario, Canada.
authors: Ben
authors: B
authors: Fielding
authors: Department of Biology, McMaster University, Hamilton, Ontario, Canada abedard@mcmaster.ca.
authors: Pierre-André
authors: PA
authors: Bédard
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D047369:Chromatin Immunoprecipitation; D064550:Death-Associated Protein Kinases / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D016755:Proto-Oncogene Proteins c-jun; D034741:RNA, Small Interfering; D018808:Transcription Factor AP-1; D015688:Oncogene Protein pp60(v-src); D064550:Death-Associated Protein Kinases
keywords: AP-1; C/EBPβ; DAPK1; JunD; transformation; v-Src