Plk1 regulates FoxM1 activity during G2-M phase

LB-221 Polo-like kinase 1 (Plk1) has essential roles in the multiple events during M phase, including mitotic entry, spindle formation, cytokinesis and mitotic exit. However, little is known about its physiological substrates and how it orchestrates several events during cell division. While searching for Plk1 binding proteins, we identified the Forkhead Box M1 (FoxM1) transcription factor as a substrate of Plk1. Recently, increasing evidence suggests that FoxM1 is a core transcription factor which regulates the G2/M transcriptional program and ensures mitotic progression, similar to the role of E2F transcription factors during the G1/S transition. The phenotypes resulting from the loss of FoxM1 expression ( such as delay at G2 phase, frequent chromosome missegregation, defects in cytokinesis and overt aneuploidy) are reminiscent of a subset of phenotypes described previously in those of Plk1. This would suggest that FoxM1 and Plk1 may function in the same and/or overlapping signaling pathway. In fact, FoxM1 exhibited a similar expression pattern as Plk1 during the cell cycle. As with other Plk1 substrates, we also identified two CDK2 phosphorylation sites on FoxM1 that are critical for the binding to the Polo-box domain (PBD) of Plk1. Furthermore, FoxM1 can be phosphorylated by Plk1 in vitro as well as in vivo and this phosphorylation occurs during G2/M phase. Phosphorylation by Plk1 resulted in the activation of FoxM1 transcriptional activity. Analysis of serial deletion mutations revealed that Plk1 phosphorylates FoxM1 at multiple sites, with the major region of phosphorylation being the c-terminal half of FoxM1. Among several potential phosphorylation sites identified by Mass spectrometry, we found two major Plk1 phosphorylation sites on FoxM1. A phospho-mutant form of FoxM1 (FoxM1-2A), in which the two major phosphorylation sites are replaced by alanines, abolished the Plk1-induced activation of FoxM1. Conversely, a phospho-mimic form of FoxM1 (FoxM1-2E), in which the two major phosphorylation sites are substituted by glutamic acid, exhibited constitutive activation. These data suggest that those two phosphorylation sites are responsible for the majority of Plk1-mediated regulation. Interestingly, silencing of FoxM1 by small interfering RNA resulted in mitotic defects, which were rescued by ectopic expression of wild-type FoxM1, but not FoxM1 with mutations in the Plk1 phosphorylation sites. These results reveal that Plk1 regulates the G2/M transcriptional program through transcriptional activation of FoxM1. Based on these findings, we propose that after initial phosphorylation by Cdks, Plk1 functions to activate FoxM1, resulting in the initiation of the G2/M transcriptional program ensuring the proper mitotic progression. In summary, these data identified a novel role of Plk1 during mitosis and show a novel mechanism by which Plk1 is involved in the control of the transcription program during cell division.