Study On The Phosporylatioin Of KIF4A In Tumor Cell

KIF4A, a member of kinesin family member4, it can binding microtubule and moving towards its plus-end. Furthermore, KIF4A can interact with many other proteins within the cell and plays crucial roles in mitosis, DNA damage repair, carcinogenesis, and neuronal survival. Some study showed that kif4a overexpression in NSCLC can be a biomarker for early prognosis, even though the exact mechanism on tumor genesis and progression is still unclear and the correlationship with clinic needs further investigation.KIF4A overexpression in tumor cells can increasing mitosis activity, enforcing proliferate ability, by contrast, it can also suppress the invasion and migration capacities. During early mitosis, KIF4A knockdown results in aneuploid by mitosis checkpoint defect through interfering chromosome congression and segregation. While on the late mitosis, cytokineis failure gives rise to multiploid. All of these increase genomic instability which is the potential initiate factor of tumorgenesis. KIF4A’s complicated functions in tumor genesis and progression indicates that it must be under a tight temporo-spatial regulation.Objective1.To identify the phosporylate site of KIF4A in tumor cells.2.To study the effects of KIF4A’s phosporylation to its function within cells.Methods1. Immunoprecipitation was preceded using rabbit anti-KIF4A multi-clone antibody to bind endogenous KIF4A. Mass spectrum based phosporylation sites enrichment was taken on the specific band cutting off after SDS-PAGE analysis.2. Comparing the results of software prediction and mass spectrum, potential site was mutated to un-phosporylate form or mimic-phosporylate form. Different forms KIF4A were transfected to Hela cells in order to study the influence on its cellular localization.3. Specific phosphorylation antibody of KIF4A was produced from immuning rabbits with specific peptide designed according to the phosphorylation site as antigen. Antibody specificity was analysised through westernblot. Antibody purification was generated through affinity Chromatography and then followed immunofluorescence analysis.4. The influences of KIF4A’s phosphorylation on its cellular fuctions were studied through rescue experiment which is transfecting exogenous isoforms to cells knocked KIF4A down previously by siRNA. Centrosomes in K562cells were extracted through density gradient centrifugation. Protein spectrum analysis was carried out on the sample after Western Blot testing in order to study the effect of KIF4A’s phosphorylation to centrosome separation.Results1. T1161site was recognized as KIF4A’s CDK1-phosphorylation site by comprehensive analysis through software prediction and mass spectrum.2. GFP-KIF4A fusion protein isoform expression constructs were successfully developed from pEGFP-KIF4A-WT plasmid. Un-phosphorylation isoform having no chromosome localization during mitosis were discovered by immunofluorescence.3. KIF4A wild type and phosphorylation isoforms could rescue early mitosis arrest resulting from KIF4A knockdown, while un-phosphorylation isoform didn’t.ConclusionKIF4A’s phosphorylation by CDK1on T1161site in early mitosis is crucial for its chromosome localization and plays a very important role in ensuring its normal functions during this phase.