The DICE-binding activity of KH domain 3 of hnRNP K is affected by c-Src-mediated tyrosine phosphorylation |
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Authors: | Messias Ana C Harnisch Christiane Ostareck-Lederer Antje Sattler Michael Ostareck Dirk H |
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Institution: | Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany. |
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Abstract: | hnRNP K and hnRNP E1/E2 are RNA-binding proteins comprised of three hnRNP K-homology (KH) domains. These proteins are involved in the translational control and stabilization of mRNAs in erythroid cells. hnRNP E1 and hnRNP K regulate the translation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA. Both proteins bind specifically to the differentiation control element (DICE) in the 3' untranslated region (3'UTR) of the r15-LOX mRNA. It has been shown that hnRNP K is a substrate of the tyrosine kinase c-Src and that tyrosine phosphorylation by c-Src inhibits the binding of hnRNP K to the DICE. Here, we investigate which of the three KH domains of hnRNP E1 and hnRNP K mediate the DICE interaction. Using RNA-binding assays, we demonstrate DICE-binding of the KH domains 1 and 3 of hnRNP E1, and KH domain 3 of hnRNP K. Furthermore, with RNA-binding assays, NMR experiments and in vitro translation studies, we show that tyrosine 458 in KH domain 3 of hnRNP K is important for the DICE interaction and we provide evidence that it is a target of c-Src. |
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Keywords: | hnRNP heteronuclear ribonucleoprotein r15-LOX reticulocyte 15-lipoxygenase DICE differentiation control element 3′UTR 3′ untranslated region GST glutathione-S-transferase NOE nuclear Overhauser effect |
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