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Study of Protein Phosphatase 2A (PP2A) Activity in LPS-Induced Tolerance Using Fluorescence-Based and Immunoprecipitation-Aided Methodology

Division of Critical Care Medicine, Department of Pediatrics and Communicable Diseases, C.S Mott Children's Hospital, University of Michigan Medical School, Ann Arbor, MI 48109, USA
Author to whom correspondence should be addressed.
Academic Editor: Ivana Vancurova
Biomolecules 2015, 5(3), 1284-1301;
Received: 29 April 2015 / Revised: 3 June 2015 / Accepted: 4 June 2015 / Published: 29 June 2015
(This article belongs to the Special Issue Transcriptional Regulation of Pro-Inflammatory Genes)
Protein phosphatase 2A (PP2A) is one of the most abundant intracellular serine/threonine (Ser/Thr) phosphatases accounting for 1% of the total cellular protein content. PP2A is comprised of a heterodimeric core enzyme and a substrate-specific regulatory subunit. Potentially, at least seventy different compositions of PP2A exist because of variable regulatory subunit binding that accounts for various activity modulating numerous cell functions. Due to the constitutive phosphatase activity present inside cells, a sensitive assay is required to detect the changes of PP2A activity under various experimental conditions. We optimized a fluorescence assay (DIFMU assay) by combining it with prior anti-PP2A immunoprecipitation to quantify PP2A-specific phosphatase activity. It is also known that prior exposure to lipopolysaccharides (LPS) induces “immune tolerance” of the cells to subsequent stimulation. Herein we report that PP2A activity is upregulated in tolerized peritoneal macrophages, corresponding to decreased TNF-α secretion upon second LPS stimulation. We further examined the role of PP2A in the tolerance effect by using PP2ACαl°xl°x;lyM-Cre conditional knockout macrophages. We found that PP2A phosphatase activity cannot be further increased by tolerance. TNF-α secretion from tolerized PP2ACαl°xl°x;lyM-Cre macrophages is higher than tolerized control macrophages. Furthermore, we showed that the increased TNF-α secretion may be due to an epigenetic transcriptionally active signature on the promoter of TNF-α gene rather than regulation of the NFκB/IκB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance. View Full-Text
Keywords: PP2A; immune tolerance; phosphatase assay PP2A; immune tolerance; phosphatase assay
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Sun, L.; Pappy, A.L., II; Pham, T.T.; Shanley, T.P. Study of Protein Phosphatase 2A (PP2A) Activity in LPS-Induced Tolerance Using Fluorescence-Based and Immunoprecipitation-Aided Methodology. Biomolecules 2015, 5, 1284-1301.

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