title: Quantitative analysis of the human spindle phosphoproteome at distinct mitotic stages.
abstract: During mitosis, phosphorylation of spindle associated proteins is a key regulatory mechanism for spindle formation, mitotic progression, and cytokinesis. In the recent past, mass spectrometry has been applied successfully to identify spindle proteomes and phosphoproteomes, but did not address their dynamics. Here, we present a quantitative comparison of spindle phosphoproteomes prepared from different mitotic stages. In total, we report the identification and SILAC based relative quantitation of 1940 unique phosphorylation sites and find that late mitosis (anaphase, telophase) is correlated with a drastic alteration in protein phosphorylation. Further statistical cluster analyses demonstrate a strong dependency of phosphorylation dynamics on kinase consensus patterns, thus, linking subgroups of identified phosphorylation sites to known key mitotic kinases. Surprisingly, we observed that during late mitosis strong dephosphorylation occurred on a significantly larger fraction of phospho-threonine than phospho-serine residues, suggesting a substrate preference of phosphatases for phospho-threonine at this stage. Taken together, our results constitute a large quantitative data resource of phosphorylation abundances at distinct mitotic stages and they provide insight into the systems properties of phosphorylation dynamics during mitosis.
authors.affiliation: Max Planck Institute of Biochemistry, Department of Cell Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Malik
authors.forename: René
authors.initials: R
authors.lastname: Lenobel
authors.forename: Anna
authors.initials: A
authors.lastname: Santamaria
authors.forename: Albert
authors.initials: A
authors.lastname: Ries
authors.forename: Erich A
authors.initials: EA
authors.lastname: Nigg
authors.forename: Roman
authors.initials: R
authors.lastname: Körner
mesh_terms: D016000:Cluster Analysis; D016384:Consensus Sequence; D003470:Culture Media; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology*; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D010768:Phosphoserine; D010769:Phosphothreonine; D010770:Phosphotransferases; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D008941:Spindle Apparatus / Q000378:metabolism*; D013997:Time Factors
chemical_list: D003470:Culture Media; D010750:Phosphoproteins; D020543:Proteome; D010769:Phosphothreonine; D010768:Phosphoserine; D010770:Phosphotransferases