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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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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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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