Abstract
The AU-rich element (ARE)-mediated mRNA-degradation activity of the RNA binding K-homology splicing regulator protein (KSRP) is regulated by phosphorylation of a serine within its N-terminal KH domain (KH1). In the cell, phosphorylation promotes the interaction of KSRP and 14-3-3ζ protein and impairs the ability of KSRP to promote the degradation of its RNA targets. Here we examine the molecular details of this mechanism. We report that phosphorylation leads to the unfolding of the structurally atypical and unstable KH1, creating a site for 14-3-3ζ binding. Using this site, 14-3-3ζ discriminates between phosphorylated and unphosphorylated KH1, driving the nuclear localization of KSRP. 14-3-3ζ –KH1 interaction regulates the mRNA-decay activity of KSRP by sequestering the protein in a separate functional pool. This study demonstrates how an mRNA-degradation pathway is connected to extracellular signaling networks through the reversible unfolding of a protein domain.
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Acknowledgements
I.D.-M. was supported by European Molecular Biology Organization (EMBO), fellowship number 240-2005. The work of R.G. is supported by a grant from the Associazione Italiana per le Ricerca sul Cancro (AIRC) and the Istituto Superiore di Sanita' (ISS), whereas P.B. is a recipient of a Senior Scholar Consultancy Grant from American Italian Cancer Foundation (AICF). The work of R.G. and P.B. is also supported by the Italian Comitato Interministerial per le Programmazione Economica (CIPE)-2007. We would like to thank J. Kleinjung and A. Pandini for the fragment-based search SCOP40 database, C. De Chiara for advice on the ARIA protocols, A. Oreggioni for his help in recording spectra, and I. Taylor for checking the oligomerization state of protein constructs by MALLS. We would like to thank K. Rittinger (MRC National Institute for Medical Research) for the gift of the GST–14-3-3ζ expression vector and for help in recording the ITC measurements and M. Lalle (Istituto Superiore di Sanita') for the gift of the difopein expression plasmid. Finally, we would like to thank P. Fletcher (MRC National Institute for Medical Research) for the synthesis of many of the KH1 peptides. All NMR spectra were recorded at the MRC Biomedical NMR centre.
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Cloning and site-specific mutagenesis was performed by D.H. Protein purification was performed by I.D.-M. and D.H. NMR experiments were performed by I.D.-M., G.K., T.A.F. and A.R. NMR data analysis was performed by I.D.-M. and M.G.-M. Structure calculations were performed by I.D.-M. CD experiments and analysis of CD data were performed by A.R. and S.M. MS analysis was performed by S.H. Phosphorylation experiments were designed and carried out by D.H. ITC experiments were performed by A.R. Subcellular localization experiments were performed by P.B. and R.G. The paper was written by I.D.-M. and A.R.
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Díaz-Moreno, I., Hollingworth, D., Frenkiel, T. et al. Phosphorylation-mediated unfolding of a KH domain regulates KSRP localization via 14-3-3 binding. Nat Struct Mol Biol 16, 238–246 (2009). https://doi.org/10.1038/nsmb.1558
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DOI: https://doi.org/10.1038/nsmb.1558
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