MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment.
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.
Marchese
Francesco P
FP
Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, London W6 8LH, United Kingdom.
Aubareda
Anna
A
Tudor
Corina
C
Saklatvala
Jeremy
J
Clark
Andrew R
AR
Dean
Jonathan L E
JL
D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D001482:Base Composition; D051546:Cyclooxygenase 2 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D051379:Mice; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D054398:Receptors, CCR4 / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D051816:Tristetraprolin / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099190:MAP-Kinase-Activated Kinase 2
D048948:14-3-3 Proteins; D051546:Cyclooxygenase 2; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D054398:Receptors, CCR4; D012260:Ribonucleases; D014157:Transcription Factors; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C515354:CCR4 protein, human; C489780:CNOT8 protein, human; C445084:mRNA deadenylase