title: Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.
abstract: The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA.
authors: pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors: Sebastian
authors: S
authors: Schulz
authors: Anke
authors: A
authors: Doller
authors: Nicole R
authors: NR
authors: Pendini
authors: Jacqueline A
authors: JA
authors: Wilce
authors: Josef
authors: J
authors: Pfeilschifter
authors: Wolfgang
authors: W
authors: Eberhardt
mesh_terms: D021581:Active Transport, Cell Nucleus; D019943:Amino Acid Substitution; D002460:Cell Line; D051959:ELAV Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D050527:Mesangial Cells / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D051959:ELAV Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D012694:Serine; D011493:Protein Kinase C
keywords: 3′UTR; 3′untranslated region; ARE; AU-rich element; CD; EMSA; HuR; HuR shuttling; MST; PKC; Phosphomimetic mutation; Posttranslational modification; Protein kinase C; RNA binding; circular dichroism; electrophoretic mobility shift assay; human antigen R; microscale thermophoresis; protein kinase C