RNAi screen in mouse astrocytes identifies phosphatases that regulate NF-κB signaling

Regulation of NF-kappa B activation is controlled by a series of kinases; however, the roles of phosphatases in regulating this pathway are poorly understood. We report a systematic RNAi screen of phosphatases that modulate NF-kappa B activity. Nineteen of 250 phosphatase genes were identified as regulators of NF-kappa B signaling in astrocytes. RNAi selectively regulates endogenous chemokine and cytokine expression. Coimmunoprecipitation identified associations of distinct protein phosphatase 2A core or holoenzymes; with the IKK, NF-kappa B, and TRAF2 complexes. Dephosphorylation of these complexes leads to modulation of NF-kappa B transcriptional activity. In contrast to IKK and NF-kappa B, TRAF2 phosphorylation has not been well elucidated. We show that the Thr117 residue in TRAF2 is phosphorylated following TNF alpha stimulation. This phosphorylation process is modulated by PP2A and is required for TRAF2 functional activity. These results provide direct evidence for TNF-induced TRAF2 phosphorylation and demonstrate that phosphorylation is regulated at multiple levels in the NF-kappa B pathway.