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Phosphorylation-mediated unfolding of a KH domain regulates KSRP localization via 14-3-3 binding

Nature Structural & Molecular Biology volume 16, pages 238–246 (2009)Cite this article

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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Figure 1: Domain structure and sequence alignment of the KSRP protein.
Figure 2: High-resolution solution structure of the KSRP KH1 domain.
Figure 3: KH1 and KH2 do not make contact.
Figure 4: KH1 phosphorylation by AKT kinase.
Figure 5: KH1 S193A and S193D mutants.
Figure 6: KH1–14-3-3ζ interaction.
Figure 7: Subcellular KSRP localization.

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References

  1. Audic, Y. & Hartley, R.S. Post-transcriptional regulation in cancer. Biol. Cell 96, 479–498 (2004).

    Article  CAS  Google Scholar 

  2. Kontoyiannis, D., Pasparakis, M., Pizarro, T.T., Cominelli, F. & Kollias, G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity 10, 387–398 (1999).

    Article  CAS  Google Scholar 

  3. Barreau, C., Paillard, L. & Osborne, H.B. AU-rich elements and associated factors: are there unifying principles? Nucleic Acids Res. 33, 7138–7150 (2006).

    Article  Google Scholar 

  4. Pullmann, R. et al. Analysis of turnover and translation regulatory RNA-binding protein expression through binding to cognate mRNAs. Mol. Cell. Biol. 27, 6265–6278 (2007).

    Article  CAS  Google Scholar 

  5. Chen, C.-Y. et al. AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell 107, 451–464 (2001).

    Article  CAS  Google Scholar 

  6. Gherzi, R. et al. A KH domain RNA binding protein, KSRP, promotes ARE-directed mRNA turnover by recruiting the degradation machinery. Mol. Cell 14, 571–583 (2004).

    Article  CAS  Google Scholar 

  7. García-Mayoral, M.F. et al. The structure of the C-terminal KH domains of KSRP reveals a non canonical motif important for mRNA degradation. Structure 15, 485–498 (2007).

    Article  Google Scholar 

  8. Briata, P. et al. p38-dependent phosphorylation of the mRNA decay-promoting factor KSRP controls the stability of select myogenic transcripts. Mol. Cell 20, 891–903 (2005).

    Article  CAS  Google Scholar 

  9. Gherzi, R. et al. The RNA-binding protein KSRP promotes decay of β-catenin mRNA and is inactivated by PI3K-AKT signaling. PLoS Biol. 5, e5 (2006).

    Article  Google Scholar 

  10. Ruggiero, T. et al. Identification of a set of KSRP target transcripts up-regulated by PI3K-AKT signaling. BMC Mol. Biol. 8, 28–43 (2007).

    Article  Google Scholar 

  11. Yaffe, M.B. et al. The structural basis for 14–3-3:phospho-peptide binding specificity. Cell 91, 961–971 (1997).

    Article  CAS  Google Scholar 

  12. Mackintosh, C. Dynamic interactions between 14–3-3 proteins and phospho-proteins regulate diverse cellular processes. Biochem. J. 381, 329–342 (2004).

    Article  CAS  Google Scholar 

  13. Aitken, A. 14–3-3 proteins: a historic overview. Semin. Cancer Biol. 16, 162–172 (2006).

    Article  CAS  Google Scholar 

  14. Gardino, A.K., Smerdon, S.J. & Yaffe, M.B. Structural determinants of 14–3-3 binding specificities and regulation of sub cellular localization of 14–3-3-ligand complexes: a comparison of the X-ray crystal structures of all human 14–3-3 isoforms. Semin. Cancer Biol. 16, 173–182 (2006).

    Article  CAS  Google Scholar 

  15. Stoecklin, G. et al. MK2-induced tristetraprolin:14–3-3 complexes prevent stress granule association and ARE-mRNA decay. EMBO J. 23, 1313–1324 (2004).

    Article  CAS  Google Scholar 

  16. Johnson, B.A., Stehn, J.R., Yaffe, M.B. & Blackwell, T.K. Cytoplasmic localization of tristetraprolin involves 14–3-3-dependent and -independent mechanisms. J. Biol. Chem. 277, 18029–18036 (2002).

    Article  CAS  Google Scholar 

  17. Musco, G. et al. The solution structure of the first KH domain of FMR1, the protein responsible for the fragile X syndrome. Nat. Struct. Biol. 4, 712–716 (1997).

    Article  CAS  Google Scholar 

  18. Baber, J.L., Levens, D., Libutti, D. & Tjandra, N. Chemical shift mapped DNA-binding sites and 15N relaxation analysis of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K. Biochemistry 39, 6022–6032 (2000).

    Article  CAS  Google Scholar 

  19. Ramos, A. et al. Role of dimerization in KH/RNA complexes: the example of Nova KH3. Biochemistry 41, 4193–4201 (2002).

    Article  CAS  Google Scholar 

  20. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  21. Camproux, A.C., Gautier, R. & Tufféry, P. A hidden Markov model derived structural alphabet for proteins. J. Mol. Biol. 339, 591–605 (2004).

    Article  CAS  Google Scholar 

  22. Pandini, A., Bonati, L., Fraternali, F. & Kleinjung, J. MinSet: a general approach to derive maximally representative database subsets by using fragment dictionaries and its application to the SCOP database. Bioinformatics 23, 515–516 (2007).

    Article  CAS  Google Scholar 

  23. Beuth, B., Pennell, S., Arnvig, K.B., Martin, S.R. & Taylor, I.A. Structure of a Mycobacterium tuberculosis NusA-RNA complex. EMBO J. 24, 3576–3587 (2005).

    Article  CAS  Google Scholar 

  24. Git, A. & Standart, N. The KH domains of Xenopus Vg1RBP mediate RNA binding and self-association. RNA 8, 1319–1333 (2002).

    Article  CAS  Google Scholar 

  25. Valverde, R., Pozdnyakova, I., Kajander, T., Venkatraman, J. & Regan, L. Fragile X mental retardation syndrome: structure of the KH1–KH2 domains of fragile X mental retardation protein. Structure 15, 1090–1098 (2007).

    Article  CAS  Google Scholar 

  26. Bernadó, P. et al. Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations. J. Biomol. NMR 29, 21–35 (2004).

    Article  Google Scholar 

  27. Lewis, H.A. et al. Sequence-specific RNA binding by a Nova KH domain: implications for paraneoplastic disease and the fragile X syndrome. Cell 100, 323–332 (2000).

    Article  CAS  Google Scholar 

  28. Schanda, P. & Brutscher, B. Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J. Am. Chem. Soc. 127, 8014–8015 (2005).

    Article  CAS  Google Scholar 

  29. Sreerama, N., Venyaminov, S.Y. & Woody, R.W. Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal. Biochem. 287, 243–251 (2000).

    Article  CAS  Google Scholar 

  30. Masters, S.C. & Fu, H. 14–3-3 proteins mediate an essential anti-apoptotic signal. J. Biol. Chem. 276, 45193–45200 (2001).

    Article  CAS  Google Scholar 

  31. Johnson, L.N. & O'Reilly, M. Control by phosphorylation. Curr. Opin. Struct. Biol. 6, 762–769 (1996).

    Article  CAS  Google Scholar 

  32. Messías, A.C., Harnisch, C., Ostareck-Lederer, A., Sattler, M. & Ostareck, D.H. The DICE-binding activity of KH domain 3 of hnRNP K is affected by c-src-mediated tyrosine phosphorylation. J. Mol. Biol. 361, 470–481 (2006).

    Article  Google Scholar 

  33. García-Mayoral, M.F., Díaz-Moreno, I., Hollingworth, D. & Ramos, A. The sequence selectivity of KSRP explains its flexibility in the recognition of the RNA targets. Nucleic Acids Res. 36, 5290–5296 (2008).

    Article  Google Scholar 

  34. Zhang, S., Xing, H. & Musli, A.J. Nuclear localization of protein kinase U-α is regulated by 14–3-3. J. Biol. Chem. 274, 24865–24872 (1999).

    Article  CAS  Google Scholar 

  35. Faul, C., Hüttelmaier, S., Oh, J., Hachet, V., Singer, R.H. & Mundel, P. Promotion of importin α-mediated nuclear import by the phosphorylation-dependent binding of cargo protein to 14–3-3. J. Cell Biol. 169, 415–424 (2005).

    Article  CAS  Google Scholar 

  36. Chou, C.F. et al. Tethering KSRP, a decay-promoting AU-rich element-binding protein, to mRNAs elicits mRNA decay. Mol. Cell. Biol. 26, 3695–3706 (2006).

    Article  CAS  Google Scholar 

  37. Lin, W.J., Duffy, A. & Chen, C.-Y. Localization of AU-rich element-containing mRNA in cytoplasmic granules containing exosome subunits. J. Biol. Chem. 282, 19958–19968 (2007).

    Article  CAS  Google Scholar 

  38. Markovtsov, V. et al. Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of poly-pyrimidine tract binding protein. Mol. Cell. Biol. 20, 7463–7479 (2000).

    Article  CAS  Google Scholar 

  39. Blichenberg, A. et al. Identification of a cis-acting dendritic targeting element in MAP2 mRNAs. J. Neurosci. 19, 8818–8829 (1999).

    Article  CAS  Google Scholar 

  40. Masino, L., Martin, S.R. & Bayley, P.M. Ligand binding and thermodynamic stability of a multidomain protein, calmodulin. Protein Sci. 9, 1519–1529 (2000).

    Article  CAS  Google Scholar 

  41. Linge, J.P., O'Donoghue, S.I. & Nilges, M. Automated asignment of ambiguous nuclear overhauser effects with ARIA. Methods Enzymol. 339, 71–90 (2001).

    Article  CAS  Google Scholar 

  42. Ye, J., Mayer, K.L., Mayer, M.R. & Stone, M.J. NMR solution structure and backbone dynamics of the CC chemokine eotaxin-3. Biochemistry 40, 7820–7831 (2001).

    Article  CAS  Google Scholar 

  43. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

    Article  CAS  Google Scholar 

  44. Bartels, C., Xia, T.-H., Billeter, M., Güntert, P. & Wüthrich, K. The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR 6, 1–10 (1995).

    Article  CAS  Google Scholar 

  45. Koradi, R., Billeter, M. & Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55 (1996).

    Article  CAS  Google Scholar 

  46. De Chiara, C. et al. The AXH domain adopts alternative folds: the solution structure of HBP1 AXH. Structure 13, 743–753 (2005).

    Article  CAS  Google Scholar 

  47. Laskowski, R.A., Rullman, J.A., MacArthur, M.W., Kaptein, R. & Thorton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    Article  CAS  Google Scholar 

  48. Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G. & Gibson, T.J. Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23, 403–405 (1998).

    Article  CAS  Google Scholar 

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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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Author notes

  1. Irene Díaz-Moreno and David Hollingworth: These authors contributed equally to the work.

Authors and Affiliations

  1. Molecular Structure Division, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

    Irene Díaz-Moreno, David Hollingworth, Steven Howell, MaríaFlor García-Mayoral & Andres Ramos

  2. MRC Biomedical NMR Centre, The Ridgeway, Mill Hill, London, NW7 1AA, UK

    Thomas A Frenkiel & Geoff Kelly

  3. Physical Biochemistry Division, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

    Stephen Martin

  4. Istituto Nazionale per la Ricerca sul Cancro, Largo Benzi Rosanna, 10, Genova, 16132, Italy

    Roberto Gherzi & Paola Briata

  5. Instituto de Bioquímica Vegetal y Fotosíntesis, US-CSIC, Avda. Americo Vespucio 49, Sevilla, 41092, Spain

    Irene Díaz-Moreno

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Contributions

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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Correspondence to Andres Ramos.

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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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