Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.
Eukaryotic cells replicate by a complex series of evolutionarily conserved events that are tightly regulated at defined stages of the cell division cycle. Progression through this cycle involves a large number of dedicated protein complexes and signaling pathways, and deregulation of this process is implicated in tumorigenesis. We applied high-resolution mass spectrometry-based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics. Co-regulated proteins and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global activation during S phase of the DNA damage response network, which was mediated by phosphorylation by ATM or ATR or DNA-dependent protein kinases. We determined site-specific stoichiometry of more than 5000 sites and found that most of the up-regulated sites phosphorylated by cyclin-dependent kinase 1 (CDK1) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells.
Olsen
Jesper V
JV
Department of Proteomics and Signal Transduction, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried near Munich, Germany.
Vermeulen
Michiel
M
Santamaria
Anna
A
Kumar
Chanchal
C
Miller
Martin L
ML
Jensen
Lars J
LJ
Gnad
Florian
F
Cox
Jürgen
J
Jensen
Thomas S
TS
Nigg
Erich A
EA
Brunak
Søren
S
Mann
Matthias
M
D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D016000:Cluster Analysis; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis; D011485:Protein Binding; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D013379:Substrate Specificity
D010750:Phosphoproteins; D020543:Proteome; D016203:CDC2 Protein Kinase; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2