Study of protein phosphatase 2a (Pp2a) activity in lps-induced tolerance using fluorescence-based and immunoprecipitation-aided methodology

Lei Sun, Adlai L. Pappy, Tiffany T. Pham, Thomas P. Shanley*

*Corresponding author for this work

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

 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 NFkB/IkB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.

Original languageEnglish (US)
Pages (from-to)1284-1301
Number of pages18
JournalBiomolecules
Volume5
Issue number3
DOIs
StatePublished - Jul 7 2015

Fingerprint

Protein Phosphatase 2
Immunoprecipitation
Fluorescence
Macrophages
Phosphoric Monoester Hydrolases
Assays
Immune Tolerance
Lipopolysaccharides
Phosphoprotein Phosphatases
Peritoneal Macrophages
Epigenomics
Genes

Keywords

  • Immune tolerance
  • PP2A
  • Phosphatase assay

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry
  • Molecular Biology

Cite this

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title = "Study of protein phosphatase 2a (Pp2a) activity in lps-induced tolerance using fluorescence-based and immunoprecipitation-aided methodology",
abstract = " 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 NFkB/IkB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.",
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Study of protein phosphatase 2a (Pp2a) activity in lps-induced tolerance using fluorescence-based and immunoprecipitation-aided methodology. / Sun, Lei; Pappy, Adlai L.; Pham, Tiffany T.; Shanley, Thomas P.

In: Biomolecules, Vol. 5, No. 3, 07.07.2015, p. 1284-1301.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Study of protein phosphatase 2a (Pp2a) activity in lps-induced tolerance using fluorescence-based and immunoprecipitation-aided methodology

AU - Sun, Lei

AU - Pappy, Adlai L.

AU - Pham, Tiffany T.

AU - Shanley, Thomas P.

PY - 2015/7/7

Y1 - 2015/7/7

N2 -  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 NFkB/IkB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.

AB -  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 NFkB/IkB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.

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