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

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

SUBMITTER: Choudhary S

PROVIDER: S-EPMC3199466 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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

Choudhary Sanjeev S Rosenblatt Kevin P KP Fang Ling L Tian Bing B Wu Zhao-Hui ZH Brasier Allan R AR

The Journal of biological chemistry 20110907 43

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-o ...[more]

PMID: 21900239

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Project description:BackgroundThe aryl hydrocarbon receptor (AhR) is a transcription factor activated by several environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and involved in carcinogenesis and various physiological processes, including immune response and endocrine functions. Characterization of kinases-related AhR transduction pathway remains an important purpose.ResultsWe performed a kinome-wide siRNA screen in human mammary MCF-7 cells to identify non redundant protein kinases implicated in the up-regulation of cytochrome P-450 (CYP) 1A1 activity, an AhR referent target, in response to TCDD exposure. To this aim, we monitored CYP1A1-related ethoxyresorufin-O-deethylase (EROD) activity and quantified cell density. This normalization was crucial since it allowed us to focus only on siRNA affecting EROD activity and discard siRNA affecting cell density. Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits. Collectively, these data fully validated the protocol and the siRNA library. Next, 22 novel candidates were identified as kinases potentially implicated in the up-regulation of CYP1A1 in response to TCDD, without alteration of cell survival or cell proliferation. The siRNA library screen gave a limited number of hits (approximately 3%). Interestingly, four of them are able to bind calmodulin among which the IP3 kinase A (ITPKA) and pregnancy up-regulated non-ubiquitously expressed CaM kinase (PNCK, also named CaMKI?). Remarkably, for both proteins, their kinase activity depends on the calmodulin binding. Involvement of ITPKA and PNCK in TCDD-mediated CYP1A1 up-regulation was further validated by screening-independent expression knock-down. PNCK was finally shown to regulate activation of CaMKI?, a CaMKI isoform previously reported to interplay with the AhR pathway.ConclusionsThese data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade. More generally, this study also highlights the interest of large scale loss-of-function screens for characterizing the molecular mechanism of action of environmental contaminants.

Dataset Information

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

Publications

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

Similar Datasets

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