Technical Report abstract
Nature Structural & Molecular Biology 15, 321 - 329 (2008)
Published online: 24 February 2008 | doi:10.1038/nsmb.1395
In situ observation of protein phosphorylation by high-resolution NMR spectroscopy
Philipp Selenko1,3, Dominique P Frueh1, Simon J Elsaesser1,3, Wilhelm Haas2, Steven P Gygi2 & Gerhard Wagner1
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.
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.
- Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.
- 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.).
Correspondence to: Philipp Selenko1,3 e-mail: selenko@fmp-berlin.de
Correspondence to: Gerhard Wagner1 e-mail: gerhard_wagner@hms.harvard.edu
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