RNAi screen to identify protein phosphatases that regulate the NF-kappaB signaling

Abstract

NF-kappaB plays a critical role in cell survival, apoptosis, and inflammatory responses. Serine/threonine-specific phosphatases (PPs) represent the second major class of enzymes that catalyze the dephosphorylation of proteins. The roles of PPs regulating NF-kappaB activities are poorly understood. Here we describe an RNAi-based screen to identify the PPs that involve in regulating NF-kappaB signaling. Thirty-four candidate PPs siRNAs were synthesized and primarily screened by NF-kappaB reporter gene assay in HeLa cells. PHLPP, one of the protein phosphatase type 2C family members (PP2C), was identified as a positive regulator of NF-kappaB signaling. Knock-down of PHLPP dramatically attenuated TNFα-stimulated NF-kappaB transcriptional activation. Knock-down of PHLPP led to enhancement of NF-kappaB/p65 nuclear import and retention, but decreased TNFα-induced phosphorylation at Ser276 on p65. This critical phosphorylation was also drastically reduced by knock-down of PKCalpha and Akt1, two important serine/threonine kinases dephosphorylated by PHLPP. The results together suggest that PHLPP-Akt-PKC may represent an important signaling loop that activates NF-kappaB/p65 signaling through critical serine phosphorylation.

Keywords

nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) / protein serine/threonine phosphatases / PH domain leucine-rich repeat protein phosphatase (PHLPP) / RNA interference

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Acknowledgements

This research was supported by the National High Technology Research and Development Program of China (863 Program) (No.2006AA02Z191), the Bureau of Science and Technology of Guangzhou, China (No. 2007Z1-E4041) and Guangzhou Economic & Technological Development District (GETDD S & T Project) (2007G-P029).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg