title: Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs.
abstract: Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3300 unique proteins during early mitotic exit, providing up to eightfold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (∼ 10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical transition.
authors.affiliation: From the ‡The Kinghorn Cancer Center, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia;
authors.forename: Rachael A
authors.initials: RA
authors.lastname: McCloy
authors.affiliation: §The Charles Perkins Center, School of Molecular Bioscience and Sydney Medical School, The University of Sydney, NSW 2006, Australia;
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Parker
authors.affiliation: From the ‡The Kinghorn Cancer Center, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia;
authors.forename: Samuel
authors.initials: S
authors.lastname: Rogers
authors.affiliation: §The Charles Perkins Center, School of Molecular Bioscience and Sydney Medical School, The University of Sydney, NSW 2006, Australia;
authors.forename: Rima
authors.initials: R
authors.lastname: Chaudhuri
authors.affiliation: From the ‡The Kinghorn Cancer Center, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia;
authors.forename: Velimir
authors.initials: V
authors.lastname: Gayevskiy
authors.affiliation: §The Charles Perkins Center, School of Molecular Bioscience and Sydney Medical School, The University of Sydney, NSW 2006, Australia;
authors.forename: Nolan J
authors.initials: NJ
authors.lastname: Hoffman
authors.affiliation: From the ‡The Kinghorn Cancer Center, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia;
authors.forename: Naveid
authors.initials: N
authors.lastname: Ali
authors.affiliation: From the ‡The Kinghorn Cancer Center, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia; ¶St. Vincent's Clinical School, Faculty of Medicine, UNSW, Darlinghurst, NSW, Australia; ‖Department of Thoracic Medicine, St Vincent's Hospital, Darlinghurst, NSW, 2010, Australia;
authors.forename: D Neil
authors.initials: DN
authors.lastname: Watkins
authors.affiliation: **Department of Biochemistry and Molecular Biology, School of Biomedical Sciences Monash University, Clatyon, VIC, 3800, Australia;
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Daly
authors.affiliation: §The Charles Perkins Center, School of Molecular Bioscience and Sydney Medical School, The University of Sydney, NSW 2006, Australia;
authors.forename: David E
authors.initials: DE
authors.lastname: James
authors.affiliation: ‡‡Equipe Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, 1919 Route de Mende, 34293 Montpellier cedex 5, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Lorca
authors.affiliation: ‡‡Equipe Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, 1919 Route de Mende, 34293 Montpellier cedex 5, France.
authors.forename: Anna
authors.initials: A
authors.lastname: Castro
authors.affiliation: ¶St. Vincent's Clinical School, Faculty of Medicine, UNSW, Darlinghurst, NSW, Australia; From the ‡The Kinghorn Cancer Center, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia; a.burgess@garvan.org.au.
authors.forename: Andrew
authors.identifier: http://orcid.org/0000-0003-4536-9226
authors.initials: A
authors.lastname: Burgess
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000705:Anaphase; D017124:Conserved Sequence; D019143:Evolution, Molecular; D006367:HeLa Cells; D006801:Humans; D008677:Metaphase; D008938:Mitosis; D008954:Models, Biological; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D010748:Phosphopeptides; D010750:Phosphoproteins; D011494:Protein Kinases