High Throughput Short Interfering RNA (siRNA) Screening of the Human Kinome Identifies Novel Kinases Controlling the Canonical Nuclear Factor-κB (NF-κB) Activation Pathway

Nuclear factor-kappa B (NF-kappa B) is an inducible cytoplasmic transcription factor that plays a role as a master regulator of airway mucosal inflammation. The prototypical ("canonical") NF-kappa B 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-kappa B 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-kappa B 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-kappa B/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-kappa B activation in the TNF-induced canonical pathway.