Abstract
Tristetraprolin (TTP) directs its target AU-rich element (ARE)-containing mRNAs for degradation by promoting removal of the poly(A) tail. The p38 MAPK pathway regulates mRNA stability via the downstream kinase MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2), which phosphorylates and prevents the mRNA-destabilizing function of TTP. We show that deadenylation of endogenous ARE-containing tumor necrosis factor mRNA is inhibited by p38 MAPK. To investigate whether phosphorylation of TTP by MK2 regulates TTP-directed deadenylation of ARE-containing mRNAs, we used a cell-free assay that reconstitutes the mechanism in vitro. We find that phosphorylation of Ser-52 and Ser-178 of TTP by MK2 results in inhibition of TTP-directed deadenylation of ARE-containing RNA. The use of 14-3-3 protein antagonists showed that regulation of TTP-directed deadenylation by MK2 is independent of 14-3-3 binding to TTP. To investigate the mechanism whereby TTP promotes deadenylation, it was necessary to identify the deadenylases involved. The carbon catabolite repressor protein (CCR)4.CCR4-associated factor (CAF)1 complex was identified as the major source of deadenylase activity in HeLa cells responsible for TTP-directed deadenylation. CAF1a and CAF1b were found to interact with TTP in an RNA-independent fashion. We find that MK2 phosphorylation reduces the ability of TTP to promote deadenylation by inhibiting the recruitment of CAF1 deadenylase in a mechanism that does not involve sequestration of TTP by 14-3-3. Cyclooxygenase-2 mRNA stability is increased in CAF1-depleted cells in which it is no longer p38 MAPK/MK2-regulated.
Highlights
The p38 MAPK pathway inhibits AU-rich element (ARE)-mediated decay allowing for dynamic control of the expression of these mRNAs [1,2,3]. p38 MAPK regulates mRNA stability via the downstream kinase MAPKAP kinase 2 (MK2), which phosphorylates [4, 5] and prevents the function [6, 7] of the mRNA-destabilizing ARE-binding protein, tristetraprolin (TTP)
We recently showed that dual control of mRNA stability by TTP and the p38 MAPK pathway is a general mechanism, which operates for many mRNAs of the inflammatory response [8]
The importance of TTP in the control of inflammatory gene expression is demonstrated by spontaneous inflammatory arthritis in TTPϪ/Ϫ mice arising mainly from increased tumor necrosis factor (TNF) production [9]. p38 MAPK regulates mRNA stability by direct phosphorylation and inactivation of another ARE-binding protein, KH domain-splicing regulatory protein [10, 11]. p38 MAPK inhibitors fail to destabilize mRNAs of the inflammatory response in macrophages from TTPϪ/Ϫ mice [8, 12]
Summary
TTP has been reported to interact with mRNA decay factors including the exosome [30], Dcp1a, Dcp, Xrn, and CCR4 [31] but not PARN [29] It is not clear which deadenylase is involved in TTP-directed deadenylation in cells. We modified an in vitro ARE-dependent and TTP-directed deadenylation assay described by Lai et al [29] to use bacterially expressed recombinant TTP This allowed the involvement of deadenylases to be determined by assaying extracts from cells depleted of different deadenylases by RNAi in the presence of a constant amount of TTP. The assay uses TNF and granulocyte/macrophage-colony stimulating factor (GM-CSF) ARE RNA substrates with 100-nt poly(A) tails Deadenylation of both of these mRNAs has been shown previously to be regulated by TTP [16, 32]. The in vitro deadenylation assay enabled us to use R18 and difopein to test the function of 14-3-3 in deadenylation and to determine a novel mechanism whereby MK2 inhibits TTPdirected deadenylation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
