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Phosphorylation‐independent recruitment of 14‐3‐3 to tristetraprolin partially impairs deadenylation by blocking Ccr4, but not Pan2, association
Author(s) -
Nunez Castrejon Ana M.,
Tun Maria,
Nava Raul,
Kraemer Laura,
Clement Sandra L.
Publication year - 2016
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.30.1_supplement.808.1
Subject(s) - phosphorylation , tristetraprolin , hek 293 cells , untranslated region , kinase , immunoprecipitation , messenger rna , microbiology and biotechnology , mutant , transfection , chemistry , rna binding protein , biology , gene , biochemistry
The regulation of mRNA stability by RNA binding proteins plays a key role in the modulation of gene expression. The RNA‐binding protein tristetraprolin (TTP) binds to AU‐rich elements (AREs) in the 3′ untranslated regions (UTRs) of transiently expressed mRNAs encoding cytokines, growth factors, and proto‐oncogenes and stimulates their decay by recruiting a number of mRNA decay enzymes including the Ccr4 and Pan2 deadenylase complexes. Phosphorylation of serines 52 and 178 of TTP by the p38‐activated MAP kinase MK2 impairs TTP's ability to stimulate mRNA decay by preventing the recruitment of these deadenylases. Phosphorylation of TTP also creates a binding site for the protein 14‐3‐3, although it is unclear whether 14‐3‐3 association is required to impair TTP function. In order to determine whether 14‐3‐3 association is sufficient to prevent deadenylase recruitment in the absence of phosphorylation, 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. The ability of Myc‐tagged wild‐type and mutant forms of TTP to bind FLAG‐tagged Ccr4, Caf1, or Pan2 deadenylases was tested in anti‐FLAG co‐immunoprecipitation assays from transiently transfected Human Embryonic Kidney (HEK) 293T cell lysates. Mutant forms of TTP that bound 14‐3‐3 independently of phosphorylation were unable to interact with the either the Ccr4 or Caf1 proteins but retained their ability to associate with Pan2 whereas forms of TTP that failed to bind 14‐3‐3 bound all three proteins. These results suggest that 14‐3‐3 association with TTP independently of phosphorylation does not fully recapitulate the effect of phosphorylation. Support or Funding Information This project was supported by a California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Grant to SLC. ANC received a stipend from the Cal Poly College of Science and Mathematics 2015 Summer Research Program. MT received a stipend and research funds from the Allan Hancock College/Cal Poly SLO Bridges to the Baccalaureate program funded by the National Institutes of Health.