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 PP2ACαlox/lox;lyM-Cre conditional knockout macrophages. We found that PP2A phosphatase activity cannot be further increased by tolerance. TNF-α secretion from tolerized PP2ACαlox/lox;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.
Highlights
Protein phosphorylation plays a key role in the regulation of many cellular functions such as cell growth and differentiation, cell death, immune responses and neurological activities [1,2,3,4]
The phosphoprotein phosphatases (PPPs) family is comprised of serine/threonine phosphatases including the most abundant protein phosphatases: PP1, phosphatase 2A (PP2A) and PP2B, as well as less abundant PP4, PP5, PP6 and PP7
We found that tolerance significantly induced PP2A activity
Summary
Protein phosphorylation plays a key role in the regulation of many cellular functions such as cell growth and differentiation, cell death, immune responses and neurological activities [1,2,3,4]. A balance between protein kinases and protein phosphatases regulates these phosphorylation-dependent responses. In addition to protein tyrosine (Tyr) phosphatases (PTPs), the protein Ser/Thr phosphatases (PSPs) compose another large protein phosphatase family. PSPs are classified into three subtypes: phosphoprotein phosphatases (PPPs), metal-dependent protein phosphatases (PPMs), and the aspartate-based phosphatases represented by FCP/SCP [5]. The PPP family is comprised of serine/threonine phosphatases including the most abundant protein phosphatases: PP1, PP2A and PP2B, as well as less abundant PP4, PP5, PP6 and PP7. PP2A is a key cellular serine-threonine phosphatase, accounting for ~1% of total cell protein content
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