Dephosphorylation of the RNA‐ Binding Protein Tristetraprolin by PP2A in LPS‐Stimulated Macrophages

Reversible post‐translational processes can regulate gene expression by rapidly changing levels of messenger RNA (mRNA), and consequently, levels of the proteins they encode. For example, the RNA‐binding protein tristetraprolin (TTP) stimulates mRNA decay by binding to adenylate‐‐uridylate‐‐rich elements (AREs) in the 3′ untranslated region of target mRNAs and recruiting mRNA decay enzymes. Various external stimuli can induce phosphorylation of TTP by the p38 MAPK‐‐activated kinase MK2. Phosphorylation of TTP inhibits the recruitment of mRNA decay factors and results in the transient stabilization of target transcripts. Dephosphorylation of TTP allows TTP to resume mRNA decay, thereby preventing the constant production of these target proteins. Previous studies have implicated the heterotrimeric phosphatase PP2A in the dephosphorylation and subsequent reactivation of TTP, but it is unknown how and when this phosphatase is targeted to TTP. In order to better characterize the TTP dephosphorylation process, we used SDS‐PAGE and western blotting to observe the relative abundance and phosphorylation of TTP in cell lysates from RAW 264.7 murine macrophages stimulated with lipopolysaccharide (LPS). TTP levels were highest approximately 12 hours after LPS induction, with dephosphorylation commencing roughly 16 hours after LPS induction. We also observed the relative abundance of components of the PP2A enzyme including the catalytic (C) and three candidate regulatory subunits (B, B′ and B″) of the heterotrimer. We found that PP2A C, B and B″ protein levels remained relatively constant throughout the time course. In contrast, levels of PP2A B′ (B56) were not detectable until 12 and 14 hours after LPS stimulation and declined shortly thereafter. The simultaneous increase of PP2A B′ abundance and TTP dephosphorylation suggests that this regulatory subunit may play a role in targeting PP2A to TTP. We are currently performing co‐immunoprecipitation assays between TTP and PP2A subunits in order to determine whether the interaction between these proteins is regulated.