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

Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, London W6 8LH, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2010; 285(36):27590-600. DOI: 10.1074/jbc.M110.136473
Source: PubMed


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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    • "As an example of PUF-based RBP engineering, we fused the PUM-HD domain to TTP, a well-studied post-transcriptional regulator. TTP binds to AU-rich elements (AREs) in the 3′-untranslated region (UTR) of the target genes and promotes mRNA degradation by recruiting a deadenylase complex [30]. TTP is also known to promote ARE-dependent gene knockdown via translation repression through cooperation with a general translation inhibitor RCK/P54 [31], though the details of this mechanism remain unknown. "
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    • "RNA was isolated using RNeasy kit (Qiagen) and cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). mRNAs were quantified by qRT-PCR using Taqman Gene expression assays and data analysed by relative quantitation (with standard curves) with normalisation to GAPDH mRNA as previously described [34]. "
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    • "MK2-mediated phosphorylation of TTP does not affect its binding to target mRNA, but inhibits the ability of TTP to recruit deadenylases to target mRNA for their degradation in cytoplasm (Carballo et al., 2001; Stoecklin et al., 2004; Clement et al., 2011). Further investigation of this demonstrated that carbon catabolite repressor protein 4-associated factor-1 (CAF1) was the major source of deadenylase activity responsible for TTP-directed deadenylation (Marchese et al., 2010). MK2 phosphorylation reduced the ability of TTP to promote deadenylation by inhibiting the recruitment of CAF1 deadenylase independently of 14-3-3. "
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