High throughput short interfering RNA (siRNA) screening of the human kinome identifies novel kinases controlling the canonical nuclear factor-κB (NF-κB) activation pathway

Sanjeev Choudhary, Kevin P. Rosenblatt, Ling Fang, Bing Tian, Zhao Hui Wu, Allan R. Brasier

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Nuclear factor-κB (NF-κB) is an inducible cytoplasmic transcription factor that plays a role as a master regulator of airway mucosal inflammation. The prototypical ("canonical") NF-κB pathway controls cytoplasmic to nuclear translocation in response to stimulation by the mononuclear cytokine, TNF. Despite intensive investigation, the spectrum of kinases involved in the canonical NF-κB pathway has not yet been systematically determined. Here we have applied a high throughput siRNA-mediated loss-of-function screening assay to identify novel kinases important in TNF-induced NF-κB signaling. Type II A549 epithelial cells stably expressing an IL-8/luciferase reporter gene optimized for high throughput siRNA format (Z′score of 0.65) and siRNAs for 636 human kinases were reverse-transfected and screened in the assay. 36 candidate genes were identified that inhibited TNF signaling with a Z score deviation of <-1.3 in replicate plates. From this group, 11 kinases were selected for independent validation, of which eight were successfully silenced. Six kinases were validated, including ATM, CDK2, -5, and -7, CALM3, MAPAKP5, and MAP3K/MEKK3. The surprising function of ATM in TNF signaling was confirmed where reduced NF-κB/RelA translocation and Ser-276 phosphorylation were seen in ATM -/- mouse embryo fibroblasts. These data indicate that ATM is a key regulatory kinase that may control global NF-κB activation in the TNF-induced canonical pathway.

Original languageEnglish (US)
Pages (from-to)37187-37195
Number of pages9
JournalJournal of Biological Chemistry
Volume286
Issue number43
DOIs
StatePublished - Oct 28 2011

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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