The p38/MK2-Driven Exchange between Tristetraprolin and HuR Regulates AU–Rich Element–Dependent Translation (original) (raw)
Figure 5
MK2-dependent competition in TNF–ARE binding of HuR and TTP in vitro and in LPS-stimulated macrophages.
A) Electrophoretic mobility shift assay (EMSA) of an ARE mRNA fragment with purified HuR and TTP and supershift of the specific bands by specific antibodies, respectively. B) In vitro phosphorylation of TTP by activated MK2 in the presence of gamma-33P-ATP. Recombinant GST-p38 was used to activate recombinant GST-MK2, which then phosphorylates the bona-fide substrate Hsp25 (as control) and TTP. Autophosphorylation of GST-p38 and GST-MK2 is also detected. C) Competition in binding of TTP and HuR to the ARE probe in the absence of MK2 activity (protein amounts in ng). Infrared images of proteins cross-linked to the ARE probe and separated in SDS-Page are shown. D) p38/MK2-dependence of the equilibrium of binding of TTP and HuR to the ARE. E,F) Titrations for the estimation of the ARE-binding affinity (represented by approximate Kd values) for CIP-dephosphorylated and p38/MK2 phosphorylated HuR (E) and TTP (F). Samples were analyzed by EMSA and by cross-linking (CL). E) HuR concentrations between 0.25–2×10−6 (as indicated) were applied to the binding assays. F) TTP concentrations between 0.25–8×10−6 were applied to the binding assays. G) Comparison of ARE-binding between identical concentrations (4×10−6 M) of dephospho- (TTP) and phospho-TTP (p38, MK2, TTP) by EMSA. H–J) HuR-RNA-IP using lysates of MK2-rescued and GFP-transfected macrophages after 1 h of LPS-stimulation. H) TNF mRNA in the HuR-IP was quantified in two independent samples. GFP-RNA-IP was analyzed as negative control. I) ß-actin mRNA in the HuR- and GFP-IP. J) TNF mRNA/ß-actin mRNA ratio in the HuR-IP of MK2-rescued and GFP-transduced macrophage cell lines. P values of the two-tailed student's t-test are given.