Messias Ana C, Harnisch Christiane, Ostareck-Lederer Antje, Sattler Michael, Ostareck Dirk H
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany.
J Mol Biol. 2006 Aug 18;361(3):470-81. doi: 10.1016/j.jmb.2006.06.025. Epub 2006 Jun 30.
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.
异质核糖核蛋白K（hnRNP K）和异质核糖核蛋白E1/E2是由三个hnRNP K同源（KH）结构域组成的RNA结合蛋白。这些蛋白质参与红细胞中mRNA的翻译控制和稳定性调节。hnRNP E1和hnRNP K调节网织红细胞15-脂氧合酶（r15-LOX）mRNA的翻译。这两种蛋白质都特异性结合r15-LOX mRNA 3'非翻译区（3'UTR）中的分化控制元件（DICE）。研究表明，hnRNP K是酪氨酸激酶c-Src的底物，c-Src介导的酪氨酸磷酸化抑制hnRNP K与DICE的结合。在此，我们研究hnRNP E1和hnRNP K的三个KH结构域中哪一个介导与DICE的相互作用。通过RNA结合试验，我们证明了hnRNP E1的KH结构域1和3以及hnRNP K的KH结构域3与DICE结合。此外，通过RNA结合试验、核磁共振实验和体外翻译研究，我们表明hnRNP K的KH结构域3中的酪氨酸458对于与DICE的相互作用很重要，并且我们提供证据表明它是c-Src的作用靶点。
