Aurora-A Induces BRCA2 Phosphorylation And Its Relationship With Cancer

Background: Breast cancer which threatens the females lives is one of the most common malignances in the world.Although it is possible to examine who has breast cancer in early phases,the survival rate of patients after surgery still has no changes.Tumor metastasis and resistances of radiotherapy or chemotherapy are the important reasons for patients' deaths,and also difficult to treat in clinic.Radiotherapy and chemotherapy have been widely applied in breast cancer treatment,and can be provided that they can decrease the risk of local recurrence and reduce the forward mortality rate.Therefore,the major problem is resistances of DNA damage and apoptosis cause by radiotherapy and chemotherapy.Finally,they will induce tumor recurrence and metastasis.So we need to find and identify new molecular markers for targeting therapy to research breast cancer mechanism and diagnose and treat it.BRCA2(Breast cancer type 2 susceptibility protein)plays an tumor suppressor role by repairing DNA damage and being involved in mitosis and cytokinesis.But the detail mechanisms have not been researched.Aurora-A(Breast cancer activated kinase,BTAK/Aurora-A)which highly expressed in tumor cells can induce tumor development by repairing DNA damage and being involved in mitosis,cytolinesis and transcription.Also,there are researches about the relationship between Aurora-A and BRCA2.Purpose: Many studies have found that some roles of BRCA2 in tumors are played with phosphorylation by other proteins.And some other studies have reported that Aurora-A can induce some proteins' phosphorylation,such as P53.Based on these researches,we assumed whether BRCA2 could be phosphorylated by Aurora-A or not.So we researched it for further.Methods and Results: This study successfully induced protein expression of Aurora-A and BRCA2 by the technology of protein expression in vitro.After the expression we built phosphorylation systems between Aurora-A and P53(positive control),Aurora-A and BRCA2.Finally,we found that BRCA2 could be phosphorylated by Aurora-A on the threonine in the closed C terminal domain of3064-3208 bp.We have done the following four researches:1.Segmented amplification of BRCA2 in vitroFirst,we used PCR to produce amplification of segmenting BRCA2 to 16 fragments with the aid of primers which designed with Not I and Sal I(these enzymes are located in vector but not in BRCA2 plasmid)respectively in its 3' and 5' terminal.Then we linked fragments and vector PGEX-4T3,which processed by restriction enzyme digestion,into recombinant plasmid.In addition,we transformed the recombinant plasmid into BL21,and induced their bulk duplication in suitable condition.After,we used IPTG to induce protein expression in BL21 in different conditions.Finally,we used western blot to detect the expression of different fragments under different conditions by GST primary antibody.Above experiments showed that our study successfully induced the expression of16 recombinant plasmids and screened the optimum conditions aim at different plasmids.2.Inducing P53 protein phosphorylation by Aurora-AThere were reports indicated that P53 can be phosphorylated by Aurora-A on ser-315.So we choose the phosphorylation between Aurora-A and P53 to be the positive control.This study always used the same technology in part 1 to build PGEX-4T3-P53 and PGEX-4T1-Aurora-A recombinant plasmids and induce their expression.Also we used western blot to detect the expression.After reading and synthesizing amount of papers,we choose one of the phosphorylation systems.Then we detected it by the phosphorylation between P53 II and Aurora-A.Then we used SDS loading buffer to stop reaction.Finally we used western blot to detect them by phospho-P53(Ser 315)and phospho-Aurora-A(Thr288)primary antibody.Above results indicated that we first induced the expression of PGEX-4T3-P53 and PGEX-4T1-Aurora-A and screened the optimum conditions.Second,the phosphorylation system had been validated by the phosphorylation between Aurora-A and P53.3.Inducing 16 BRCA2-fragment proteins phosphorylation by Aurora-ABased on the efficiency of the phosphorylation system,we induced the phosphorylation between Aurora-A and 16 BRCA2-frgment respectively.First,we induce phosphorylation between Aurora-A and BRCA2-fragment and use BRCA2 without phosphorylation to be the negative control.In the same way,we used SDS loading buffer to stop the reaction.Then we use phospho-serine and phospho-threonine primary antibody to detect by western blot.Results showed that BRCA2 can be phosphorylated by Aurora-A on thr.4.Segemented amplification of PGEX-4T3-BRCA2-fragment 15 in vitro,and their phosphorylation by Aurora-ABased on the result that BRCA2 can be phosphorylated by Aurora-A on thr between 2950-3208 bp,we segmented this fragment again.According to the protein structure and the percentage of AT-GC,we designed primes between them.Then we used same methods to build recombinant plasmids and express their proteins.In addition,we induced these proteins under phosphorylated by Aurora-A.We found that there is a phosphorylation on PGEX-4T3-BRCA2-frgment 15-2,and this means that we shortened the region of the phosphorylation in 3064-3208 bp closed C terminal.Conclusion: Our study indicated that BRCA2 can be phosphorylated by Aurora-A in its domain of 2950-3208.It meant that it occurred on thr in C terminal.Therefore,we can form a hypothesis that one of thrs in closed C terminal on BRCA2 can affect the functions of BRCA2,such as the combination with RAD51.And it will have the impact on tumor emergence and development.Maybe it can be a target for tumor therapy.But because of the uncertainty of the precise site,it needs further study.