Phosphorylation Of BCKDK At Y246 By Src Promotes Metastasis Of Colorectal Caner

Background:Metastasis is the major cause for the mortality of colorectal patients,and meanwhile targeted therapy and biomarker research for metastatic colorectal cancer(m CRC)meet the bottleneck.Branched-chain amino acids(BCAAs)catabolism is tightly associated with tumor development.The branched-chain?-keto acid dehydrogenase kinase(BCKDK)is a vital regulation enzyme of BCAAs catabolism.Our previous study showed that BCKDK promotes colorectal cancer(CRC)tumorigenesis through activating the MAPK signaling pathway.However,the role of BCKDK in m CRC is still unknown.Objective:1.To determine the role of BCKDK in m CRC.2.To elucidate the regulation mechanism of upstream kinase Src on BCKDK.3.To explore the relationship between BCKDK activity regulation and CRC metastasis,and provide novel ideas for targeted therapy of m CRC.Methods:1.The m RNA expression level of BCKDK in CRC and corresponding normal tissues was analyzed in Oncomine database.2.The protein level of BCKDK was evaluated in the tissue microarrays(TMA)of 114CRC patients by immunohistochemical(IHC)staining.IHC results and clinical information of CRC patients were combined to determine whether BCKDK expression was associated with clinical prognosis,and was achieved by using Kaplan-Maier analysis.3.The expression of BCKDK in seven CRC cell lines was detected,and BCKDK was knockdown in HCT116 and SW620 cell lines.Wound healing and Transwell assays were performed in BCKDK knockdown cells.Expression of EMT markers(E-cadherin,Vimentin and N-cadherin)was detected by Western blot.4.The wound healing and Transwell assays were performed to investigated the effects of BCAAs stimulation on the migration and invasion of HCT116 and SW620 cells.5.BCKDK knockdown(sh BCKDK#2)and control(sh MOCK)HCT116 cells were injected into BALB/C nu-nu nude mice(n=10 per group)by tail vein,respectively,to investigate the number of metastatic nodules in lung surface of mice.6.In vitro kinase assay was applied to investigate whether upstream Src kinase can directly phosphorylates BCKDK.Net Phos 3.1 software was used to predict possible tyrosine phosphorylation sites on BCKDK.Two high-scored BCKDK peptides(Y151and Y246)were obtained and employed as substrates for active Src.7.P-BCKDK(Y246)antibody was prepared commercially,and purified by our laboratory.8.HEK293T cells were transfected with p CMV-BCKDK-WT-Flag and p CMV-BCKDK-Y246F-Flag.The cell lysates were immunoprecipitated with Flag antibody,and the immunoprecipitate were incubated with active Src.Western blot was performed using purified p-BCKDK(Y246)antibody.9.Immunofluorescence assays were performed to detect the colocalization of Src and BCKDK in HCT116 and HEK293T cells.Co-immunoprecipitation(Co-IP)assays were performed to test whether Src could bind with BCKDK.10.Increasing amounts of pc DNA₄-Src-His were transiently transfected into HEK293T cells,phosphorylated BCKDK was determined by Western blot with p-BCKDK(Y246)antibody.Pc DNA₄-Src-His and p CMV-BCKDK-WT-Flag were single or co-transfected into HEK293T cells,phosphorylation of BCKDK at Y246 was detected by Western blot.11.Phosphorylation of BCKDK at Y246 was detected in HCT116,Lo Vo and SW620cell lines under increasing EGF processing time.12.The expression of known BCKDK downstream molecules,p-BCKDHA(S293)and p-MEK1/2(S221),were detected in the following cells:Src high-expressed CRC cell lines(HT29 and SW620)treated with dasatinib,Src knockdown stable cell lines(HT29-sh Src and SW620-sh Src),as well as Src WT(Src^+/+)and knockout(Src^-/-)MEFs cells.13.Src^+/+,Src^-/-and transfected-HEK293T(transfected with p CMV empty vector,BCKDK-WT or BCKDK-Y246F)cells we treated with cycloheximide(CHX)to forbid de novo protein synthesis,and the remaining BCKDK protein level was investigated.14.Flag-ubiquitin,Src-His,BCKDK-WT or BCKDK-Y246F plasmid were co-transfected into HEK293T cells,the cell lysates were immunoprecipitated with BCKDK antibody,and the ubiquitination level of BCKDK was measured by Western blot.15.BCKDK-WT and BCKDK-Y246F overexpression stable cell lines were obtained in HCT15 and HT29 cells.Transwell assays and Western blot were conducted to elevate the metastatic phenotypes and expression of EMT markers of BCKDK overexpression stable cell lines,respectively.16.The expression level of p-BCKDK(Y246)in CRC tissues was detected by IHC,Kaplan-Meier survival analysis was performed to determine whether p-BCKDK(Y246)expression was associated with prognosis of CRC patients.17.BCKDK knockdown and SILAC-based phosphoproteomics analysis were combined to gain comprehensive insight into the BCKDK signaling network,especially signaling involved in cancer cell metastasis.Results:1.The m RNA level of BCKDK was significantly higher in colorectal carcinoma and rectum adenocarcinoma in comparison with corresponding normal tissues.2.BCKDK protein level was upregulated in metastatic CRC tissues(n=40)compared with non-metastatic patient tissues(n=74).CRC patients with elevated BCKDK expression had significantly worse overall survival(OS).3.Directly supplementary of BCAAs couldn't accelerate the migration and invasion of CRC cells.4.Knockdown of BCKDK attenuated the migration,invasion and EMT of CRC cells.5.BCKDK promoted CRC lung metastasis in vivo.6.Src phosphorylated BCKDK at Y246 site in vitro.7.The p-BCKDK(Y246)antibody was available after purification.8.The results of Western blot using p-BCKDK(Y246)antibody indicated Src could phosphorylate BCKDK-WT,whereas mutation in BCKDK at Y246 eliminated the phosphorylation effect.9.The immunofluorescence showed that Src(red)partially colocalized with BCKDK(green).The co-immunoprecipitation(co-IP)assays demonstrated that Src bound with BCKDK.10.Src phosphorylated BCKDK in a dose-dependent manner under EGF treatment.11.P-BCKDK(Y246)expression could be detected in HCT116,Lo Vo and SW620 cell lines upon EGF stimulation.12.Intracellular Src inhibition decreased the phosphorylation of BCKDK and its downstream targets.In other words,phosphorylation of BCKDK at Y246 by Src enhanced the activity of BCKDK by promoting phosphorylation of its downstream molecules.13.Src prolonged BCKDK protein half-life(t_1/2).14.Src enhanced BCKDK stability by protecting BCKDK from ubiquitination degradation.15.The migration and invasion were remarkably upregulated in BCKDK-WT cells compared to control and BCKDK-Y246F cells.BCKDK-WT cells exerted an dramatical promotion effect on the expression of N-cadherin and Vimentin,BCKDK-Y246F cells hardly or moderately upregulated N-cadherin and Vimentin expression.16.P-BCKDK(Y246)was upregulated in m CRC tissues and linked to negative prognosis of CRC patients.17.SILAC-based phosphoproteomics approach revealed the strong potential of BCKDK in regulating multiple cellular responses,including cell proliferation,differentiation,survival and metastasis et al.Conclusion:1.BCKDK promoted CRC metastasis.2.Src phosphorylated BCKDK at Y246 site in vitro and ex vivo.Importantly,phosphorylation of BCKDK by Src enhanced the activity and stability of BCKDK.3.Phosphorylation of BCKDK at Y246 by Src promoted migration,invasion and EMT of CRC cells.In summary,our study illustrates the significance role of Src/BCKDK axis in human CRC and provides a promising novel target for m CRC targeted therapy.