FIG. 10.
Determination of phosphorylation sites of HuR targeted by PKCδ by an in vitro PKC assay. (A) For the in vitro PKC assay, 2 μg of bacterially expressed and histidine-tagged HuR was incubated with 5 μg of recombinant PKCδ in a cell-free phosphorylation assay with [γ-32P]ATP in the presence (+) or absence (−) of 100 μM EGTA. Loading of equal protein amounts was ascertained by Coomassie blue staining (data not shown). Radioactive bands detected by autoradiography of a representative gel migrate at 97 kDa and represent autophosphorylated PKCδ. (B) Schematic representation of the HuR protein and positions of putative conserved PKC phosphorylation sites (numbers indicate positions of amino acids). For generation of HuR single point mutations (HuRΔ1, HuRΔ2, HuRΔ3, and HuRΔ4) and the double mutations HuRΔ1/2 and HuRΔ2/3, serines at the indicated positions (black circles) were substituted for with alanine (left panel). In vitro PKC phosphorylation in the absence (vehicle) or presence of either wild-type HuR or the indicated point-mutated HuR proteins and recombinant PKCδ was assessed in the absence of EGTA. The addition of equal amounts of recombinant HuR was ascertained by Coomassie blue staining (input HuR) (bottom panel). The data shown are representative of three independent experiments giving similar results. (C) Flag-HuR was detected by immunofluorescence 24 h after transfection of hMC with pCMV-Flag plasmids containing either full-length wild-type HuR (HuR) or full-length HuR bearing a double mutation in serines 221 and 318 (HuRΔ2/3). Images show Flag-HuR (red) and DAPI (4′,6′-diamidino-2-phenylindole) (blue) in hMC either left untreated (vehicle) or stimulated for 2 h with 100 nM AngII (+AngII). Data are representative of two independent experiments giving similar results.