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In situ observation of protein phosphorylation by high-resolution NMR spectroscopy

Nature Structural & Molecular Biology volume 15, pages 321–329 (2008)Cite this article

Abstract

Although the biological significance of protein phosphorylation in cellular signaling is widely appreciated, methods to directly detect these post-translational modifications in situ are lacking. Here we introduce the application of high-resolution NMR spectroscopy for observing de novo protein phosphorylation in vitro and in Xenopus laevis egg extracts and whole live oocyte cells. We found that the stepwise modification of adjacent casein kinase 2 (CK2) substrate sites within the viral SV40 large T antigen regulatory region proceeded in a defined order and through intermediate substrate release. This kinase mechanism contrasts with a more intuitive mode of CK2 action in which the kinase would remain substrate bound to perform both modification reactions without intermediate substrate release. For cellular signaling pathways, the transient availability of partially modified CK2 substrates could exert important switch-like regulatory functions.

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Figure 1: Multiple sequence alignment of CK2 substrates containing tandem phosphorylation sites.
Figure 2: In vitro phosphorylation of XT111–132GB1 by CK2.
Figure 3: NMR analyses of mutant XT111–132GB1 substrates.
Figure 4: Time-resolved phosphorylation of XT107–132GB1 by CK2.
Figure 5: De novo phosphorylation of CK2 substrates in Xenopus egg extracts.
Figure 6: In vivo phosphorylation of XT111–132GB1 in Xenopus laevis oocytes.
Figure 7: Modeled interactions of XT111–132GB1 with the catalytic subunit of CK2.

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Acknowledgements

We are most grateful to J. Ruderman and to members of her group, especially E. Chung and M. Lai, for continuous support in all aspects of our Xenopus work. We thank K. Niefind and O.G. Issinger for providing the PDB coordinates of the peptide substrate CK2α model that we used as the starting structure for building the SV40-CK2α models. We also thank J. Sun for help with XPLOR to calculate the final models. P.S. gratefully acknowledges funding by the Human Science Frontier Program Organization (long-term fellowship LT00686/2004-C) and by the Max Kade Foundation. This work was also funded by grant GM47467 of the US National Institutes of Health to G.W.

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Author notes

  1. Philipp Selenko & Simon J Elsaesser

    Present address: Present addresses: Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany (P.S.) and Rockefeller University, 1230 York Avenue, New York, New York 10065, USA (S.J.E.).,

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, 02115, Massachusetts, USA

    Philipp Selenko, Dominique P Frueh, Simon J Elsaesser & Gerhard Wagner

  2. Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, 02115, Massachusetts, USA

    Wilhelm Haas & Steven P Gygi

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  1. Philipp Selenko
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Contributions

P.S. conceived the project, devised and performed the biochemical and NMR experiments and wrote the manuscript. D.P.F. performed NMR experiments and wrote the manuscript. S.J.E. performed biochemical experiments and read and approved the manuscript and the conclusions drawn therein. W.H. performed MS experiments and approved the manuscript and the conclusions drawn therein. S.P.G. and G.W. read and approved the manuscript and the conclusions drawn therein.

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Correspondence to Philipp Selenko or Gerhard Wagner.

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Selenko, P., Frueh, D., Elsaesser, S. et al. In situ observation of protein phosphorylation by high-resolution NMR spectroscopy. Nat Struct Mol Biol 15, 321–329 (2008). https://doi.org/10.1038/nsmb.1395

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