Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.
Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.
Olsen
Jesper V
JV
Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.
Blagoev
Blagoy
B
Gnad
Florian
F
Macek
Boris
B
Kumar
Chanchal
C
Mortensen
Peter
P
Mann
Matthias
M
D001665:Binding Sites; D016208:Databases, Factual; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D010455:Peptides / Q000032:analysis / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000032:analysis; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis; D015398:Signal Transduction
D009363:Neoplasm Proteins; D010455:Peptides; D020543:Proteome; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor