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Global phosphoproteomic mapping of early mitotic exit in human cells identifies novel motifs that determine substrate dephosphorylation


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 complete understanding of the breadth of phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed an unbiased, global mass spectrometry-based phosphoproteomic screen to analyze the dephosphorylation events that occur during early mitotic exit. Using this method we identified and quantified the modification of 11,954 phosphosites on 2,823 proteins during early mitotic exit, providing up to 6 fold greater resolution than previous studies. More than 2,800 phosphorylation sites have not previously been annotated, while approximately 40% of all phosphorylation sites matched the minimal consensus motif for one of the major mitotic kinases (Cdk1, MAPK, GSK3, Plk1, Aurora, PKA and CK2). 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.

Type Journal
Authors McCloy, R.; Parker, B.L.; Rogers, S.; Chaudhuri, R.; Gayevskiy, V.; Hoffman, N.J.; Ali, N.; Watkins, D.N.; Daly, R.J.; James, D.E.; Lorca, T.; Castro, A.; Burgess, A.
Published Date 2015-06-08
Published Volume 14
Published Issue 8
Published Pages 2194-2122
Status Published in-print
URL link to publisher's version
OpenAccess link to author's accepted manuscript version