MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment.
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.
Article: Novel roles of the CCR4–NOT complex[Show abstract] [Hide abstract]
ABSTRACT: The CCR4–NOT complex is a multi-subunit protein complex evolutionarily conserved across eukaryotes which regulates several aspects of gene expression. A fascinating model is emerging in which this complex acts as a regulation platform, controlling gene products ‘from birth to death’ through the coordination of different cellular machineries involved in diverse cellular functions. Recently the CCR4–NOT functions have been extended to the control of the innate immune response through the regulation of interferon signaling. Thus, a more comprehensive picture of how CCR4–NOT allows the rapid adaptation of cells to external stress, from transcription to mRNA and protein decay, is presented and discussed here. Overall, CCR4–NOT permits the efficient and rapid adaptation of cellular gene expression in response to changes in environmental conditions and stimuli.For further resources related to this article, please visit the WIREs website.Conflict of interest: The authors have declared no conflicts of interest for this article.WIREs RNA 07/2014; 5(6). · 6.15 Impact Factor
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ABSTRACT: Tissue factor (TF, CD142) is a 47 kDa transmembrane cell surface glycoprotein which triggers the extrinsic coagulation cascade and links thrombosis with inflammation. Although macrophage TF expression is known to be regulated at the RNA level, very little is known about the mechanisms involved. Poly(ADP-ribose)-polymerase (PARP)-14 belongs to a family of intracellular proteins that generate ADP-ribose post-translational adducts. Functional screening of PARP-14 deficient macrophages mice revealed that PARP-14 deficiency leads to increased tissue factor (TF) expression and functional activity in macrophages following challenge with bacterial lipopolysaccharide. This was related to an increase in TF mRNA stability. Ribonucleoprotein complex immunoprecipitation and biotinylated RNA pull-down assays demonstrated that PARP-14 forms a complex with the mRNA-destabilizing protein tristetraprolin (TTP) and a conserved AU-rich element in the TF mRNA 3' untranslated region. TF mRNA regulation by PARP-14 was selective, as TNFα mRNA, which is also regulated by TTP, was not altered in PARP-14 deficient macrophages. Consistent with the in vitro data, TF expression and TF activity, but not TNFα expression, were increased in Parp14(-/-) mice in vivo. Our study provides a novel mechanism for the post-transcriptional regulation of TF expression, indicating that this is selectively regulated by PARP-14.Blood 10/2014; · 9.78 Impact Factor
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ABSTRACT: Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3'-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3'-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.Nucleic Acids Research 08/2014; · 8.81 Impact Factor