Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs

Entry into mitosis is driven by the phosphorylation of thousands of proteins on multiple sites, triggering extensive cellular rearrangements including chromosome condensation, microtubule reorganization, and nuclear envelope breakdown (1). Many of these protein phosphorylation events are induced by cyclin dependent kinase 1 (CDK1)1, which is considered the master regulator of mitosis (2). However, several recent large-scale quantitative phosphoproteomic studies have highlighted the important role of other kinases, such as Polo like kinase 1 (PLK1), Aurora A and B, in driving many of these mitotic events (3–5). For cells to exit mitosis, the phosphorylation sites modified by these kinases must be dephosphorylated (6). Furthermore, the order of their removal must be different to that of mitotic entry, in order to create the unique mitotic exit events that produce two identical daughter cells (7, 8). The current model of how cells exit mitosis is based on the coordinated degradation and dephosphorylation of proteins. Protein degradation is driven by the anaphase promoting complex (APC) and its associated co-factors CDC20 and CDH1, which co-ordinate the ordered degradation of several key regulatory proteins, including cyclin B and securin (9, 10). Protein degradation removes proteins and hence their phosphorylation status, ensuring that mitotic exit continues in one direction and does not reverse (11, 12). However, currently only ∼170 proteins have been found to be targeted for degradation during mitotic exit (13), which although likely to be a significant underestimation, is only well short of the 5000+ proteins phosphorylated during mitosis (14). Therefore, during exit a substantial number of proteins must be dephosphorylated by phosphatases in preparation for the next G1 phase. For many years phosphatases were thought to constantly dephosphorylate substrates. However, recent discoveries have shown they are in fact highly regulated, and must first be inhibited to permit mitotic entry, and then reactivated for mitotic exit (15, 16). For the highly ordered events of mitotic exit to occur correctly, the dephosphorylation of substrates must occur in a rigid, timely, and ordered fashion. Failure to do this results in a catastrophic failure of mitosis (17, 18). This suggests that phosphatases must dictate an order of dephosphorylation for the correct timing of specific mitotic exit events. This concept is supported by recent results in budding yeast where the well characterized mitotic exit phosphatase Cdc14 (19) drives ordered dephosphorylation during mitotic exit (8). Evidence from higher-order mammalian, Xenopus and Drosophila models indicates that both phosphatase PP2A (20–22) and PP1 (23, 24) are required for mitotic exit, whereas Cdc14 appears to be dispensable (reviewed in (25)). Yet despite many of these recent advancements, we still do not fully understand the mechanisms controlling the global phosphorylation changes that occur during the initial early stages of mitotic exit. Are certain substrates preferentially dephosphorylated, and if so how do phosphatases recognize and specifically dephosphorylate these substrates? To answer these questions we undertook a global, unbiased phosphoproteomic approach to characterize the full repertoire of phosphorylation changes that occur as human cells begin exiting mitosis.