Abstract
We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the 15N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D 1H-15N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.
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28 October 2015
In the version of this article initially published, Equation 11 was incorrect. This sign within the brackets was 'plus' and it should have been 'minus'. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank P. Schmieder and M. Beerbaum for excellent maintenance of NMR infrastructure. We also thank F. Cordier for many insightful discussions, G. Lippens for expert advice and all members of the Selenko laboratory for carefully reading the manuscript and providing helpful comments. F.-X.T. was supported by a grant from the Association pour la Recherche sur le Cancer (ARC). P.S. acknowledges support by an Emmy Noether research grant (SE1-1/1794) by the Deutsche Forschungsgemeinschaft (DFG).
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F.-X.T., H.M.R., S.L., A.B., R.T., M.S. and P.S. devised and executed the experiments. Specifically, F.-X.T., H.M.R. and M.S. prepared cell extracts and measured in-extract phosphorylation reactions; F.-X.T., S.L., A.B. and R.T. performed in vitro phosphorylation measurements; and F.-X.T., H.M.R. and M.S. performed western blotting. F.-X.T., H.M.R. and P.S. prepared figures and wrote the manuscript. All authors carefully read the manuscript and approved of the conclusions drawn therein.
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Theillet, FX., Rose, H., Liokatis, S. et al. Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts. Nat Protoc 8, 1416–1432 (2013). https://doi.org/10.1038/nprot.2013.083
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DOI: https://doi.org/10.1038/nprot.2013.083
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