C-Abl Regulates The G2/M Transition Through Inhibiting The Activity Of Cdc25C

Abl is one of the best conserved branches of the tyrosine kinases.The Ablfamily proteins are composed of two members,c-Abl and Arg(Abl-related gene).Each member contains an SH3-SH2-TK domain cassette in N-terminal and actin-binding domain in C-terminal.c-Abl and Arg are more than 90% identical in SH3-SH2-TK domain.c-Abl is involved in the many physiological and biochemistry processes including cell proliferation,oxidative stress,DNA damage responses,cytoskeletal reorganization,the development of lymphocytes and cell migration.Dual specificity phosphatase Cdc25 originally discovered in yeast can dephosphorylate phospho-threonine and phospho-tyrosine.In humans,Dual specificity phosphatase Cdc25 family is composed of Cdc25 A,B and C.Cdc25 C facilitates the G2/M transition through activating the Cdc2-CyclinB complex.By co-immunoprecipitation and immunoblotting we further validated the interaction between c-Abl and Cdc25 C,which was required for the activity of c-Abl kinase.What's more,c-Abl can phosphorylate Cdc25 C at Y165 site.The phosphorylation of Cdc25 C S216 promotes the binding with 14-3-3 protein,thus sequestrating Cdc25 C in cytoplasm and delaying the G2/M transition.It was discovered that there was no apparently difference between Flag-Cdc25C/WTand Flag-Cdc25C/Y165 F about the phosphorylation of S216 and the binding with 14-3-3.That implied that the Cdc25 C Y165's phosphorylation did not affect the S216's phosphorylation and the cellular effects caused by the S216's phosphorylation.We observed that the phosphorylated Cdc2 Y15 increased,when c-Abl was overexpressed.Cdc2pY15 was constant when co-overexpression of Cdc25 C Y165F and c-Abl.That suggested that c-Abl could regulate the phosphorylation of Cdc2 Y15 through phosphorylating Cdc25 C.Flag-Cdc25C/WT and Flag-Cdc25C/Y165 F overexpressed in 293 FT cells were purified by co-immunoprecipitation and affinity replacement,then phosphatase activity of the purified proteins was analyzed in vitro.The results showed that tyrosine kinase c-Abl could inhibit the phosphatase obviously.To find out the effect on cell cycle caused by the Y165's phosphorylation,we knockout the Cdc25 C gene in HeLa using Crispr/Cas9 system,and recover the expression of Cdc25C/WT and Cdc25C/Y165 F.The stable strains were screened through the stress of puromycin and hygromycin,and the knockout effect was detected by Western blotting.Finally we obtained the stable expressive GFP-Cdc25C/WT and GFP-Cdc25C/Y165 F strains.There was apparently different shape between GFP-Cdc25C/WT strains and GFP-Cdc25C/Y165 F strains.GFP-Cdc25C/WT strains were familiar with GFP-vector control strains,with the shape of polygon and better adherence.However GFP-Cdc25C/Y165 F strains were rather small and round and a part of cells were floated in DMEM as the density of cells increased.The cells were synchronized to G2 phase and collected at definite time after released from RO3306.We observed that the pH3 S10 of GFP-Cdc25C/Y165F-1 and Y165F-2 strains achieved the peak at 1 h,however,the pH3 S10 of GFP-Cdc25C/WT continuously increased in 2 hours.The same results were obtained by immunofluorescence experiment.These data indicated that the phosphorylation defect of Cdc25 C Y165F could maintain rather high phosphatase activity,so Cdc25 C could activate Cdc2-Cyclin B complex immediately and promote G2/M transition once RO3306 was removed.The GFP-Cdc25C/WT,YF-1,YF-2 strains were synchronized to M phase by Nocodazole and collected at definite time after released from Nocodazole.The cell cycle analyzed by flow cytometry showed that their mitosis process was uniform.That indicated the phosphorylation of Cdc25 C Y165 by c-Abl only affected G2/M transition,not Mitosis progress.We also noticed that Cdc25 C Y165F mutant strains had more polyploid cells than the wild type.This suggested that Cdc25C/WT could play important roles in maintaining the stability of genome.IR could cause DNA damage and further activate ATM/ATR-Chk1/Chk2-Cdc25 C signaling,resulting in G2/M arrest.The Cdc25 C Y165F-1 and Y165F-2 strains could pass the G2/M earlier after exposed to IR.These results also demonstrated that the cells bypassed G2 checkpoint due to higher activity of Cdc25 C Y165F mutant.In summary,our research showed that 1)c-Abl could interact with Cdc25 C and phosphorylate the Y165 site.2)The phosphorylation of Cdc25 C Y165 could inhibit the Cdc25 C phosphatase activity.3)c-Abl was involved in the regulation of G2/M transition through phosphorylating Cdc25 C.Some of the human cancers where Cdc25 overexpression has been reported are breast,ovarian,pancreatic,colorectal,prostate,and non-Hodgkin lymphoma.The Cdc25 C Y165F mutants could earlier pass G2 checkpoint when respond to DNA damage,which may impair the integrity and stability of genome.This suggested us that the c-Abl-Cdc25 C signaling closely related to carcinogenesis.