C-Abl Tyrosine Kinase-mediated FoxM1Phosphorylation Plays Important Role In Mitosis Regulation

Non-receptor tyrosine kinase c-Abl is widely expressed in mammalian tissuesand plays key roles in cell proliferation, cell cycle regulation, cell apoptosis, DNAdamages repair, and oxidative stress. In normal physiological conditions, c-Abl isauto-inhibited with low kinase activity. Once activated by cytokines, oxidative stress,or DNA damage, c-Abl kinase with high activity participates in a wide range ofcellular regulations dependent on its subcellular localization. c-Abl is consisted withseveral functional domains, including Src Homology2(SH2), Src Homology3(SH3), kinase domain, nuclear localization signal (NLS) and nuclear export signal(NES). SH3domain specifically recognizes and binds with proline-rich motif (PXXP)and SH2domain recognizes and binds with phosphorylated tyrosine motif (pYXXP).Both of them is responsible for c-Abl kinase activity regulation. c-Abl kinase couldalso shuttle between nucleus and cytoplasm with the help of NLS and NES.Transcription factor FoxM1belongs to FOX family, which characterized bywinged helix in its DNA binding domains. FoxM1regulates transcription andexpression of particular genes dependent on cell cycle, and is involves in theregulation of cell proliferation, cell cycle, DNA damage repair, embryo developmentand tumorgenesis. Both c-Abl and FoxM1has been implicated in the similar cellularfunctions, in which a potential relationship between them will be uncovered. In thisstudy, we will focus on the interaction of c-Abl and FoxM1, c-Abl-mediated FoxM1phosphorylation modification, stability regulation, ubiquitination-inducedproteasomal degradation, and the effect on mitosis.Analyzed by immunoprecipitation and immunoblotting, the interaction betweenin vivo c-Abl and FoxM1was observed in MCF-7cells. The interaction of ectopicallyexpressed Flag-FoxM1and Myc-c-Abl was also observed in293FT cells, which wasnot depend on c-Abl kinase activity. Moreover, c-Abl kinase bind with FoxM1by itsSH2and SH3domain, and the binding of SH2domain was much more stronger thanSH3. Since SH2specifically recognizes and binds with the phosphorylated tyrosinemotif, it suggested that FoxM1could be a new phosphorylation substrate of c-Abltyrosine kinase.Further, it was confirmed that FoxM1could be phosphorylated by c-Abl kinase,which mainly happen in mitosis. LC-MS/MS analysis indicated that FoxM1wasphosphorylated by c-Abl at Y129, Y272, Y317, Y377and Y590. By amino acid sitemutation, we found that the phosphorylation of all mutants was decreased by differentdegree. The observation that the phosphorylation of Y272F and Y590F mutant wasless than10%of wild type FoxM1indicated that Y272and Y590were the mainphosphorylation sites on FoxM1.Also, the expression of c-Abl kinase could remarkably enhance cellular FoxM1level and prolong the half-life of FoxM1in293FT cells. Comparing with the wild type MEFs, the half-life of FoxM1in c-abl-/-arg-/-MEFs was also shortened. Further,the ubiquitination of FoxM1was analyzed, and the result showed that theubiquitination of FoxM1was remarkably inhibited by c-Abl kinase activity. TheY272/590F double mutant demonstrated very strong ubiquitin modification. Thesefindings suggested that c-Abl kinase-mediated tyrosine phosphorylation could protectFoxM1from the ubiquitin-proteasomal degradation. Cdh1is a component of APC/CE3ubiquitin ligase responsible for the recruitment of ubquitination substrates. Indepth, it was found that the binding of Cdh1with FoxM1Y272/590F mutants wasstrongly reinforced, compared with wild-type FoxM1. This result was coincident withthe high ubiquitin level of Y272/590F mutants, and proposed a mechanism of FoxM1stability regulation.Phosphorylation of FoxM1by c-Abl may play important role in cell cycleregulation, since FoxM1is an important cell cycle dependent transcription factor. Itwas observed that the cells expressing FoxM1Y590F mutant could pass through theM-phase more rapidly than the cells expressing the wild type FoxM1, and thedegradation of Cyclin B1was also accelerated by Y590F mutant expression. Theseresults demonstrated that c-Abl-mediated phosphorylated of FoxM1could affect cellcycle progression. However, the mechanism of the regulation still need to beuncovered.In summary, we found that1) c-Abl could interact with FoxM1by its SH2andSH3domain.2) c-Abl kinase-mediated phosphorylation of FoxM1mainly happenedin M-phase.3) There were five tyrosine phosphorylation sites on FoxM1(Y129, Y272,Y317, Y377and Y590), among of which, Y272and Y590were the mainphosphorylation sites.4) c-Abl could increase the stability of FoxM1by prolongingthe half-life of FoxM1.5) The ubiquitination of FoxM1was remarkably inhibited byc-Abl kinase activity though the inhibition of the interaction between FoxM1andCdh1E3ligase recruiter.6) The cells expressing FoxM1Y590F could pass throughthe M-phase more rapidly than the cells expressing the wild type FoxM1, and thedegradation of Cyclin B1was also accelerated by Y590F mutant expression, whichindicated that the Y590F mutant was more activate than the wild type.Our studies firstly confirmed the interaction between c-Abl and FoxM1, andc-Abl-mediated FoxM1tyrosine phosphorylation. Moreover, we identified fivetyrosine phosphorylation sites on FoxM1, which was never reported previously. Onone hand, c-Abl mediated FoxM1tyrosine phosphorylation could enhance cellularFoxM1level by protecting FoxM1from ubiquitin-proteasomal degradation. On theother hand, the transcriptional activity of FoxM1was somehow inhibited by tyrosinephosphorylation. The dual role of c-Abl kinase on FoxM1may contribute to thehomeostasis regulation of cell cycle progress.