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. 2010 Sep 3;285(36):27590-600.
doi: 10.1074/jbc.M110.136473. Epub 2010 Jul 1.

MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment

Francesco P Marchese  1 , Anna Aubareda, Corina Tudor, Jeremy Saklatvala, Andrew R Clark, Jonathan L E Dean
Affiliations

MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment

Francesco P Marchese et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
p38 MAPK regulates the poly(A) tail length of endogenous TNF mRNA. A and B, RAW 264.7 cells were treated with LPS (10 ng ml−1) for 2 h, and then actinomycin D (Act D; 10 μg ml−1) was added together with vehicle (0.1% dimethyl sulfoxide) or 1 μm SB202190 (SB). Cells were harvested at the time intervals shown, and RNA was extracted. A, 10 μg RNA was incubated with TNF 1246 (lanes 2–9) or TNF 1246 oligodeoxynucleotide and oligo(dT) (lanes 1 and 10) and cleaved with RNase H. The samples were Northern blotted with an antisense riboprobe against the TNF 3′-UTR. B, Northern blot of full-length TNF mRNA. GAPDH is shown as a loading control.
FIGURE 2.
FIGURE 2.
TTP promotes ARE-dependent deadenylation in vitro. A, in vitro deadenylation of 32P-labeled TNF ARE or TNF ARE antisense RNA substrates were incubated in the presence of HeLa S100 (5 μg) and 100 ng GST or GST-TTP at 37 °C for the times indicated. Representative phosphorimage of urea-PAGE of 32P-labeled reaction products is shown. Graphs show mean poly(A)100 expressed as a percentage of t = 0 ± S.E. from three independent experiments. Where not shown, error bars are smaller than the symbols. The positions of polyadenylated substrate (poly(A)100) and deadenylated product (poly(A)0(*)) are indicated. B, as for A but with GM-CSF ARE or GM-CSF ARE mut RNA substrates.
FIGURE 3.
FIGURE 3.
Phosphorylation of TTP by MK2 inhibits deadenylation. MK2 was immunoprecipitated from lysates of HeLa cells stimulated with IL-1α and used to phosphorylate recombinant GST-TTP in vitro. Phosphorylation reactions were performed for different times (30, 60, and 120 min) at 30 °C, and a nonimmune antibody (N.I.) served as a control. A, phosphorylated GST-TTP assayed by in vitro deadenylation assay for 60 min at 37 °C. B, phosphorimage of GST-TTP or GST phosphorylation by immunoprecipitated MK2 or nonimmune control (N.I.) for different times in the presence of [γ-32P]ATP. C, graph showing correlation between GST-TTP phosphorylation by MK2 (% max incorp. of 32P) and inhibition of deadenylation.
FIGURE 4.
FIGURE 4.
Phosphorylation of TTP by MK2 does not affect ARE binding by TTP. A, 32P end-labeled GM-CSF ARE RNA probe (0.1 pmol) was incubated with different concentrations of either unphosphorylated TTP or TTP phosphorylated by recombinant active MK2 for 30 min at 30 °C. B, graph of bound RNA against [TTP] and best fit with the Hill equation from three independent experiments. Where not shown, error bars are smaller than the symbols. C, 32P end-labeled GM-CSF ARE was incubated with TTP (25 nm) or GST (25 nm) in the presence or absence of increasing amounts (1, 10, and 20×) of cold GM-CSF ARE (self) or GM-CSF ARE mut (non-self) RNA competitors. Results are representative of three experiments. RNA·protein complexes were resolved by electrophoresis and visualized using a phosphorimaging device. The free probe and the TTP·RNA complexes are indicated.
FIGURE 5.
FIGURE 5.
14-3-3 binding does not mediate the effect of MK2 on TTP-directed deadenylation. A, GST-TTP (WT or S52A/S178A) was phosphorylated with MK2 as in Fig. 4 or left unphosphorylated and used in a GST pulldown assay. S100 extracts from HeLa cells were incubated with recombinant GST-TTP or GST (negative control) immobilized on glutathione-Sepharose in the presence or absence of R18 or difopein. Pulled down proteins were resolved by SDS-PAGE and Western blotted for 14-3-3 and TTP. Results are representative of three experiments. B, in vitro deadenylation assay using unphosphorylated and phosphorylated GST-TTP (wild-type and S52A/S178A) in the presence or absence of R18 (5 μm) or difopein (1 μm). Graphs of mean (±S.E.) for three independent experiments are shown.
FIGURE 6.
FIGURE 6.
Depletion of CCR4a inhibits TTP-directed deadenylation. HeLa cells were left untransfected (Untr), transfected with a scramble (Scr) control double-stranded oligoribonucleotide or with siRNAs targeting CCR4a or CCR4b, separately or in combination. In vitro deadenylation assays were performed in the presence of GST-TTP as shown in Fig. 2. A, CCR4a and CCR4b mRNA knockdown efficiencies were determined by Q-RT-PCR. Graphs of mean (±S.D.) mRNA determined by Q-RT-PCR of RNA from knockdown cells. B, Graphs show mean poly(A)100 expressed as a percentage of t = 0 ± S.D. from two independent knockdown experiments performed in duplicate. Where not shown, error bars are smaller than the symbols. Significance was determined by two-tailed unpaired t test. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
FIGURE 7.
FIGURE 7.
Depletion of CAF1a and CAF1b inhibits TTP-directed deadenylation. CAF1a or CAF1b expression was suppressed, separately or in combination (CAF1a+CAF1b) in HeLa cells by RNAi as shown in Fig. 6. A, CAF1a and CAF1b mRNA knockdown efficiencies were determined by Q-RT-PCR as in Fig. 6. B, CAF1a and CAF1b knockdown assessed by Western blot for CAF1a and CAF1b. C, in vitro deadenylation assay and graphs as in Fig. 6. Untr, untransfected; Scr, scramble.
FIGURE 8.
FIGURE 8.
Depletion of CCR4 inhibits TTP-directed deadenylation and formation of 5′-AMP. A, cells were transfected with scramble or CCR4a and CCR4b together, cells were lysed, and extracts were assayed in the presence of GST-TTP using an [α-32P]ATP-labeled RNA substrate. Two portions of the same gel are shown. B, same as A, but GM-CSF ARE or GM-CSF ARE mut RNA substrates were incubated in the presence or absence of GST-TTP (100 ng) and untransfected cell extracts for the times indicated. The position of the 5′-AMP product of the deadenylation reaction is indicated.
FIGURE 9.
FIGURE 9.
Recruitment of CAF1 to TTP is inhibited by MK2 phosphorylation and 14-3-3 independent. A–D, GST-TTP or GST alone was bound to glutathione -Sepharose 4B beads in presence of HeLa cell lysates. A, pulled down material probed for PAN2, PARN, or CAF1a. B, GST pulldown assay of GST-TTP (wild-type or S52A/S178A mutant) phosphorylated by MK2 in vitro or left unphosphorylated as in Fig. 4, in the presence or absence of 150 units of benzonase. C, a graph of mean (±S.E.) for three independent experiments of Caf1a protein quantified by densitometry. D, GST pulldown assay of wild-type GST-TTP phosphorylated by MK2 or left unphosphorylated, in the presence of 150 units of benzonase (Benz.) and in the presence or absence of 1 μm difopein.
FIGURE 10.
FIGURE 10.
CAF1 is required for regulation of COX-2 mRNA stability by p38 MAPK/MK2. A and B, HeLa cells were transfected with scramble or CAF1a and CAF1b siRNAs as in Fig. 6. Cells were then treated with IL-1 (20 ng/ml) for 90 min (A) and then treated with actinomycin D (Act D) alone or together with 1 μm SB202190 (SB) for the times indicated (B). A, graph shows COX-2/GAPDH mRNA normalized to scramble (IL-1). B, plot of % of t = 0 COX-2/GAPDH mRNA. A and B show mean and S.E. from three independent experiments.
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