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

doi:10.1074/jbc.m111.224923

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.

Highlights

Eukaryotic tissues respond to signals in their extracellular environment through the induction of long term phenotypic plasticity
Similar qualitative effects on TNF-induced TNFAIP3/A20 expression was observed in HEK293 cells, with the exception of CDK2 and MAP3K12, which did not affect TNFAIP3/A20 expression in HEK293 cells. These results suggest that the role of ataxia telangiectasia mutated (ATM), MAP3K12, PKC␨, CALM-3, and CDK7, -5, and -2 in TNF signaling is not specific to A549 cells
We standardized and applied an HT-siRNA screen to identify new kinase candidates that control the canonical nuclear factor-␬B (NF-␬B) pathway; we further sought to validate a subset of the identified kinases

Summary

EXPERIMENTAL PROCEDURES

Materials—The Stealth RNAi human kinase library, in a 96-well microtiter plate format (eight master plates), which targets 636 human kinase genes, was purchased from Invitrogen. Each plate consisted of three negative-universal siRNA controls (Invitrogen) containing low, medium, and high GC content. For siRNA screening, 25 ␮l of combined, diluted RNAis were robotically (Biomek FXp, Beckman, Brea, CA) aliquoted into a 96-well plate (Nunc, Rochester, NY), mixed with 25 ␮l of diluted transfection reagent (0.3 ␮l/well of Siquest reagent, Mirus, Madison, WI), and reverse transfected by dispensing A549-Luc stable cells (10,000 cells in 100 ␮l) into each well using the Titertek, multidrop 384 cell dispenser (effective concentration of 40 nM for each RNAi from the three duplexes for each target). Luciferase Assay—Twenty ␮l of cell lysate from each well in the transfection plates was transferred and mixed with 80 ␮l of luciferin reagent for measurement of relative luminescence (96-well plate; Nunc). Quantification of changes in gene expression was calculated using the ⌬⌬Ct method and unstimulated cells as the calibrator and normalized to GAPDH [25]

RESULTS

Average Z score

Accession number

DISCUSSION