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Phosphorylation‐independent recruitment of 14‐3‐3 alters tristetraprolin function (748.3)
Author(s) -
Kraemer Laura,
Clement Sandra
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.748.3
Subject(s) - tristetraprolin , phosphorylation , hek 293 cells , dephosphorylation , untranslated region , immunoprecipitation , messenger rna , microbiology and biotechnology , rna binding protein , transfection , kinase , chemistry , biology , phosphatase , biochemistry , gene
Cell signaling pathways influence mRNA turnover rates through reversible post‐translational modifications that alter the activity of a number of mRNA binding proteins. Tristetraprolin (TTP) is a zinc‐finger RNA binding protein that binds to AU‐rich elements (AREs) in the 3’ untranslated regions (UTRs) of transiently expressed messages encoding cytokines and pro‐inflammatory proteins. Unphosphorylated TTP causes rapid mRNA turnover by recruiting mRNA decay enzymes, but phosphorylation of TTP at serines 52 and 178 by the p38 MAPK activated kinase MK2 prevents its ability to do so. Phosphorylation also leads to the recruitment of 14‐3‐3, is associated with alterations in the localization of TTP, and may protect TTP from dephosphorylation and destruction by the proteasome. However, because mutations that destroy 14‐3‐3 binding sites in TTP also prevent its phosphorylation, it is unclear whether 14‐3‐3 binding is required to inhibit TTP function. To better understand the role of 14‐3‐3 in the inhibition of TTP we replaced serines 52 and 178 in TTP via site‐directed mutagenesis with either a peptide sequence known to bind 14‐3‐3 independently of phosphorylation or with an altered version of this peptide sequence that does not. We tested the ability of these mutant forms of TTP to recruit 14‐3‐3 using co‐immunoprecipitation assays from transiently transfected Human Embryonic Kidney (HEK) 293T cell lysates. We observed that protein levels for mutant forms of TTP that failed to associate with 14‐3‐3 were consistently lower than versions of TTP that retained the ability to do so, supporting the hypothesis that 14‐3‐3 may protect TTP from destruction by the proteasome. We also observed that recruitment of 14‐3‐3 was sufficient to alter localization of TTP from the nucleus to the cytoplasm. Experiments are currently underway to determine whether 14‐3‐3 association is sufficient to alter the recruitment of mRNA decay enzymes and the protein phosphatase PP2A.