In situ observation of protein phosphorylation by high-resolution NMR spectroscopy.
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.
Selenko
Philipp
P
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA. selenko@fmp-berlin.de
Frueh
Dominique P
DP
Elsaesser
Simon J
SJ
Haas
Wilhelm
W
Gygi
Steven P
SP
Wagner
Gerhard
G
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D047390:Casein Kinase II / Q000378:metabolism; D002457:Cell Extracts; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010063:Ovum; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013997:Time Factors; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D002457:Cell Extracts; C034284:IgG Fc-binding protein, Streptococcus; D050505:Mutant Proteins; D029867:Xenopus Proteins; D010768:Phosphoserine; D047390:Casein Kinase II