Calmodulin Regulates The Stability Of Nucleocytoplasmic Protein TBC1D3 And Its Biological Significance In Human Breast Cancer Cells

ObjectiveTBC1D3?also referred to as prostate cancer gene 17,PRC 17?was identified as a hominoid-specific gene,which widely expressed in human tissues and overexpressed in prostate,breast,bladder and pancreatic cancer as well as in myelodysplastic syndrome?MDS?.Ectopic expression of TBC1D3 confers tumorigenicity to mouse NIH 3T3 embryonic fibroblast cells,indicating that TBC1D3 functions as an oncogene.Moreover,TBC1D3 inhibits the ubiquitination of epidermal growth factor receptor?EGFR?and insulin receptor substrate-1?IRS-1?,and their subsequent degradation,thereby enhancing EGF and insulin signaling and consequential cell proliferation.Nevertheless,growth factor?GF?signaling promotes SCF-FBXW8 E3 ubiquitin ligases CUL7-mediated TBC1D3 ubiquitination and proteasomal degradation.Camodulin?CaM?has been demonstrated that regulates stability of a body of proteins,such as estrogen receptor-a?ER-a?and TRE17/USP6.Furthermore,our preliminary experiment indicated that CaM inhibits the degradation of TBC1D3,and then enhances its stability.Therefore,to explore the mechanisms by which CaM regulates the stability of TBC1D3 may be beneficial to elucidate the role of CaM and TBC1D3 in tumor development and progression,and to provide new clues for the diagnosis and prevention of tumor.Methods:1.In order to study the effect of CaM on the degradation of TBC1D3,MCF-7 and BT-549cells were transfected w:ith GST-CaM for ectopic expression or treated with CaM antagonist W7 to inhibit function of endogenous CaM,then the role of CaM in the process of TBC1D3's ubiquitination and degradation were observed by western blot and co-immunoprecipitation.And then the impact of CaM on the subcellular location of TBC1D3's degradation was observed by extracting and analyzing cytoplasmic and nuclear protein.2.In order to investigate whether the interaction between CaM and TBC1D3 inhibits the ubiquitination and degradation of TBC1D3,MCF-7 cells were co-transfected with Flag-TBC1D3 and GST Vector or GST-CaM plasmids and performed the co-immunoprecipitation with anti-GST antibody;then revised co-immunoprecipitation was performed used anti-Flag antibody in MCF-7 cells co-transfected with GST-CaM and Flag Vector or Flag-TBC1D3 plasmids.And then,the interaction between CaM and TBC1D3 was observed by immunoprecipitation with anti-Flag antibody and anti-GST antibody treated with or not Ca²⁺ chelator EGTA in MCF-7 and BT-549 cells,respectively.Furthermore,TBC1D3 deletion mutants Flag-TBC1D3?? 157-171?and Flag-TBC1D3?? 303-312?were constructed by over-lap PCR,and they were all co-transfected with GST-CaM into MCF-7 cells,as well as Flag-TBC1D3.Then cells were lysed and performed the co-immunoprecipitation with anti-GST antibody;then revised co-immunoprecipitation was performed in MCF-7 cells co-transfected with GST-CaM and Flag Vector or Flag-TBC1D3 plasmids3.In order to explore the function of interacting motif between TBC1D3,TBC1D3 internal point mutant Flag-TBC1D3?Y163A-T165A?and Flag-TBC1D3?K166R?were constructed by over-lap PCR.Then Flag-TBC1D3?Flag-TBC1D3??157-171?and point mutants were transfected into MCF-7 cells and their ubiquitination and degradation level were with detected by Western Blot and CO-IP.Finally,Flag-TBC1D3,point mutants were co-transfected with GST-CaM into MCF-7 cells and were performed immunoprecipitation with anti-Flag antibody to perform to detect their interaction with CaM.4.In order to detect the effect of TBC1D3 on cell migration,Flag vector and Flag-TBC1D3 plasmids were transfected into MCF-7 and BT-549 cells,then were observed by cell scratch and transwell migration assay.As asking for the mechanisms by which TBC1D3 affects cell migration,MCF-7 cells were respectively transfected with Flag vector and Flag-TBC1D3 plasmids,and then were starved with serum-free medium for 24 hours.The activity of MMP-9 in culture medium was detected by gelatin zymography,while the mRNA and protein levels of MMP-9 in cell lysates were measured by real-time fluorescence quantitative PCR and western blot simultaneously.Finally,MCF-7 cells were co-transfected with GST-CaM to enhance the stability of TBC1D3 and observe the impacts of enhanced TBC1D3's stability on the cell migration,MMP-9 activity and protein level.Results:1.Western blot analysis showed that 10%FCS induced the degradation of TBC1D3,while ectopic expression of GST-CaM inhibited TBC1D3's degradation,indicating that CaM could inhibit the degradation of TBC1D3.When the function of endogenous CaM was inhibited by W7,TBC1D3 degraded much obviously by 10%FCS stimulation,indicating that endogenous CaM could inhibit TBC1D3's degradation.The results in immunoprecipitation experiments showed that 10%FCS stimulation could induce the ubiquitination of TBC1D3,however,over-expression of CaM significantly reduce the TBC1D3's ubiquitination,suggesting that CaM could decrease the ubiquitination level of TBC1D3.The cytoplasmic and nuclear protein extraction showed that TBC1D3 degradation induced by 10%FCS in MCF-7 and BT-549 cells occurred both in the cytoplasm and the nucleus.What's more,there's much degradation of nuclear TBC1D3 in MCF-7cells,while much decrease of cytoplasmic TBC1D3 in BT-549 cells.Moreover,over-expression of CaM significantly reduces the TBC1D3's degradation both in the cytoplasm and the nucleus.2.Immunoprecipitation was performed with anti-GST antibody and the results showed that TBC1D3 binding band was detected in GST-CaM group but not GST control group,indicating that TBC1D3 interacted with CaM.When the immunoprecipitation was performed with anti-Flag antibody,the CaM binding band was detected in Flag-TBC1D3 group but not Flag control group.However,when Ca²⁺ was chelated by EGTA,the interaction between TBC1D3 and CaM was abolished both in co-immunoprecipitation with anti-Flag antibody and anti-GST antibody,suggesting that the interaction between CaM and TBC1D3 in a La²⁺manner.TBC1D3 deletion mutants Flag-TBC1D3??157-171?and Flag-TBC1D3?A303-312?constructed by Over-lap PCR were correctly sequenced and the results co-immunoprecipitated with anti-Flag antibody or anti-GST antibody showed that deletion mutants TBC1D3?A 157-171?abolished the interaction between TBC1D3 and CaM,suggesting that the CaM-interacting site in TBC1D3 resides at or near amino acids 157-171.3.TBC1D3 point mutants Flag-TBC1D3?Y163A-T165A?and Flag-TBC1D3?K166R?constructed by Over-lap PCR were correctly sequenced.Western blot analysis showed that 10%FCS stimulation induces the degradation of TBC1D3?Y163A-T165A?,but not TBC1D3?A157-171?and TBC1D3?K166R?.Immunoprecipitation experiments with anti Flag antibody was performed,the results showed that significant ubiquitin was detected in Flag-TBC1D3?Y163A-T165A?and Flag-TBC1D3,while hardly in Flag-TBC1D3?K166R?.Moreover,CaM binds with TBC1D3 and TBC1D3?K166R?,but not Flag control and TBC1D3?Y163A-T165A?,suggesting that CaM interacts with TBC1D3 and inhibits GF signaling-induced ubiquitination at K166 and subsequent degradation of the oncoprotein in human breast cancer cells.4.The results of cell scratches and transwell migration showed that the migrated number of MCF-7 and BT-549 cells transfected with Flag-TBC1D3 was much more than that of the cells transfected Flag vector.Real-time fluorescence quantitative PCR showed that TBC1D3 could up-regulate the level of MMP-9 mRNA;Western blot displayed that TBC1D3 enhances the expression of MMP-9;Gelatin zymogram experiments showed that TBC13 enhances the activity of MMP-9,suggesting that TBC1D3 promotes the migration of human breast cancer cells in a manner involving the expression and activation of MMP-9.Moreover,TBC13 up-regulates the level and activity of MMP-9 and induces cell migration were all enhanced by ectopic expressed CaM,suggesting that CaM enhances the TBC1D3-induced migration of human breast cancer cells by a mechanism involving the expression and activation of MMP-9.Conclusion:1.CaM enhances the stability of TBC1D3 both in the cytoplasm and the nucleus.2.CaM interacts with TBC1D3 at or near amino acids 157-171 resides in a Ca²⁺ dependent manner.3.The lysine residue 166 within the CaM-interacting motifs of TBC1D3 was the actual site for the GF signaling-induced ubiquitination.4.CaM enhances the TBClD3-induced migration of human breast cancer cells by a mechanism involving the expression and activation of MMP-9.