High throughput siRNA Screening of the Human Kinome Identifies Novel Kinases Controlling the Canonical NF-kB 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 (HT-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 HT-siRNA format (Z prime 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 8 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-kB/RelA translocation and Ser 276 phosphorylation was seen in ATM(-/-)MEFs. These data indicate that ATM is a key regulatory kinase that may control global NF-κB activation in the TNF-induced canonical pathway.

The article is published online in the Journal of Biological Chemistry and is free to access.