Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.
Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.
Daub
Henrik
H
Cell Signaling Group, Department of Molecular Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. daub@biochem.mpg.de
Olsen
Jesper V
JV
Bairlein
Michaela
M
Gnad
Florian
F
Oppermann
Felix S
FS
Körner
Roman
R
Greff
Zoltán
Z
Kéri
György
G
Stemmann
Olaf
O
Mann
Matthias
M
D000595:Amino Acid Sequence; D002453:Cell Cycle; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D016196:S Phase; D013379:Substrate Specificity
D010748:Phosphopeptides; D010750:Phosphoproteins; D010770:Phosphotransferases