C-Abl Regulates FoxM1 Stability And Transcriptional Function By Tyrosine Phosphorylation

The non-receptor tyrosine kinases c-Abl,was originally identified as the cellular homolog of the v-abl oncogene of the Abelson murine leukemia virus.c-Abl is ubiquitously expressed in mammalian tissues,involving in the regulation of cell proliferation,cell differentiation,tumorigenesis,DNA damage repair,cytoskeletal organization,apoptosis and respond to oxidative stress,via binding to and phosphorylation of its substrates.c-abl has a related gene arg,they share highly homology structure and overlapping functions.Transcription factor Fox M1 belongs to FOX family,mainly expresses in proliferation and Differentiated cells,plays key roles in cell proliferation,cell cycle progress,DNA damage repair,tumorigenesis,apoptosis,embryogenesis and organogenesis,via regulating genes expression.Since the relevance roles of c-Abl and FoxM1 playing in physiological functions such as cell cycle regulation,tumorigenesis and DNA damage repair,and c-Abl could regulate numerous transcription factors includes p53,NF-?B and C/EBP?according to previous reports,we investigate the mechanism and biological significance of c-Abl phosphorylate FoxM1.Our previous data showed that exogenous expressed c-Abl could associate with and phosphorylate Fox M1,and the phosphorylation sites were identified by LC-MS/MS analysis(they were Y129,Y272,Y317,Y362,Y575).We continued to investigate the relationship between c-Abl and Fox M1,to further study the molecular mechanism and biological significance,include ubiquitination modification,expression and transcription activity regulation,eventually the influence on cell cycle progress and apoptosis.In this study,immunoprecipitation,immunoblotting,immunofluorescence and other techniques demonstrated an endocellular interaction between c-Abl and FoxM1,which mainly locate in nuclear and happen in mitosis;we also demonstrated that endogenous FoxM1 was phosphorylated by using a tyrosine phosphorylation antibody,and the phosphorylation weaken after cells were treated with c-Abl specific inhibitor STI571.Next,we found a decline protein level of FoxM1 after c-abl/arg were knock-down or knock-out,and FoxM1 expression was down-regulated after cells were treated with STI571,implying that FoxM1 expression was depend on c-Abl kinase activity.Cells were treated with CHX,a protein synthesis inhibitor,to investigated the half-life of Fox M1,we found that the FoxM1 half-life was shortened after c-Abl/Arg knock-out,which reminded that c-Abl/Arg could increase Fox M1 protein stability.We imagined that phosphorylation of Fox M1 affect its ubiquitination-induced proteasome degradation;so we treated U2 OS cells with STI571,or STI571 combination with proteasome inhibitor MG132.It was found that the STI571 treatment remarkably enhanced FoxM1 ubiquitination and down-regulated its protein levels,whereas MG132 treatment rescued its expression,implying that c-Abl could up-regulate Fox M1 expression via inhibiting its degradation.Further,we investigated the ubiquitination modification and expression of FoxM1 mutants,found that Y272 F,Y57F and Y272-575 F mutants exhibited an increase ubiquitination,remarkably decrease protein expression,consistent with a stronger reinforced binding with E3 ubiquitin ligase component CDH1,suggested the phosphorylation at sites Y272 and Y575 stabilized Fox M1 protein;meanwhile,Y317 F and Y362 F mutants exhibited an decrease protein levels,consistence with an weaken binding to CHD1,implying phosphorylation at sites Y317 and Y362 might facilitate FoxM1 degradation.FoxM1 plays a key role in cell cycle progress via regulating numerous genes expression,including Cyclin B1,Cyclin D1,CDC25 A,Plk1,CENP-F,Aurora-A and Aurora-B,etc.We therefore examined whether regulation of FoxM1 by c-Abl affected its transcriptional activity.It was confirmed that the expression of FoxM1 and its target genes Cyclin B1,Cyclin G2,Plk1 were remarkably down-regulated after c-Abl/Arg were knock-down,further,cells treated with STI571 also exhibited a decrease transcript levels and protein levels of genes Cyclin B1,Plk1 and CENP-F,suggested that regulation of Fox M1 by c-Abl affected its transcriptional activity.Meanwhile,by cell synchronization,flow cytometry and other assays,we confirmed that STI571 treatment could arrest cell cycle progress via inhibiting expression of FoxM1 target genes include Cyclin B1,Plk1 and Aurora-B.FoxM1 could act as an inhibitor of apoptosis in tumor cells,we therefore investigated whether regulation of Fox M1 by c-Abl affected its function in apoptosis.Hela cells were treated with Doxorubicin(a chemotherapy drugs),or doxorubicin combination with STI571,then the Cleaved caspase3 and Cleaved PARP-1 expression were analyzed by immunoblotting,we found that STI571 treatment increased apoptosis induced by doxorubicin,suggested that STI571 treatment down-regulated FoxM1 expression via inhibiting c-Abl kinase activity,thus sensitizes cells to apoptosis.Our study confirmed FoxM1 was regulated by c-Abl,and many studies demonstrated that numerous transcriptional factor such as c-My,NF-?B,C/EBP?were substrates of c-Abl,implying that c-Abl might be implicated in genes transcript regulation.Therefore,total RNA prepared from MCF-7 and MCF-7 KD(c-abl/arg Knock-Down)cells were subjected to RNA-seq analysis.The results showed that a total of 1034 genes were significantly regulated by c-Abl/Arg,of which 635 were down-regulated and 399 were up-regulated in c-abl/arg knockdown cells.To further understand the potential cellular function and associated pathways of these genes,we performed GO term and KEGG pathway analyses.Data showed they were involved in numerous signaling pathway and biological process,of which mainly related to c-Abl/Arg as previous reports.Our results supported the hypothesis that c-Abl/Arg participate in many signaling pathway via regulation of genes transcript.In summary,we demonstrated that endogenous c-Abl associated with and phosphorylated FoxM1;phosphorylation of FoxM1 inhibited its ubiquitination-induced proteasome degradation,which increased its protein stability and up-regulate its expression;regulation of Fox M1 by c-Abl affected cell cycle progression and apoptosis;c-Abl/Arg played a role in genes transcript regulation.Our study provide a deeper insight into the function of c-Abl and FoxM1,especially provide essential theoretical basis for the research of their function in cell cycle regulation and cancer cells apoptosis.