title: High throughput short interfering RNA (siRNA) screening of the human kinome identifies novel kinases controlling the canonical nuclear factor-κB (NF-κB) activation pathway.
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.
authors: Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555, USA. sachoudh@utmb.edu
authors: Sanjeev
authors: S
authors: Choudhary
authors: Kevin P
authors: KP
authors: Rosenblatt
authors: Ling
authors: L
authors: Fang
authors: Bing
authors: B
authors: Tian
authors: Zhao-Hui
authors: ZH
authors: Wu
authors: Allan R
authors: AR
authors: Brasier
mesh_terms: D000818:Animals; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: C498143:RELA protein, human; D034741:RNA, Small Interfering; C498144:Rela protein, mouse; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D011494:Protein Kinases