Phosphorylation Regulation Of Methionine Adenosyl-Transferase Alpha 1 And Its Role In Liver Cancer

ObjectiveLiver cancer is one of the most common malignant tumors in China,and its exhibit an extremely poor prognosis due to postsurgical recurrence and metastases.Our team has demonstrated that methionine adenyltransferase α1(MATα1,the protein encoded by MAT1A)was expressed in normal liver and was downregulated in liver cancer.MATα1overexpression represses the progression of liver cancer.To understand MATα1 tumor suppressive function,we compared MATα1 interactome in normal versus cancerous livers and identified the 14-3-3ζ(the protein encoded by YWHAZ).Here we examined the roles and mechanisms of interaction of between 14-3-3ζ and MATα1 and provides a new therapeutic target for the therapy of liver cancer.Methods:(1)Co-immunoprecipitation and mass spectrometry examined protein-protein interactions.(2)A series of His-tagged MATα1 phosphorylation sites mutants were created to identify which site of MATα1 responsible for the 14-3-3ζinteraction.(3)Immunofluorescence assays were performed to examine the subcellular localization of MATα1 protein through co-transfection with14-3-3ζ and MATα1 or MATα1 phosphorylation site mutant plasmids.(4)silencing AKT2 expression affect the interaction of 14-3-3ζwith MATα1 by Co-immunoprecipitation.(5)The effect of 14-3-3 protein small molecule inhibitor BV02 treatment on the interaction of MATα1 with 14-3-3ζ were examined by Co-immunoprecipitation.The effect of BV02 on the proliferation,invasion and migration of liver cancer cells was examined by Brd U,Transwell and wound healing assay.BV02 treatment inhibited OKER cell growth in vivo using a syngeneic model.(6)Western blot and q RT-PCR assays were performed to detect the effect of overexpression and si RNA knockdown 14-3-3ζ on the levels of MATα1 protein and m RNA,and effect of overexpression and si RNA knockdown MATα1 on the levels of 14-3-3ζ protein and m RNA.(7)Dual luciferase reporter gene assay,chromatin immunoprecipitation and Electrophoretic Mobility Shift Assay were performed to investigate the mechanism of reciprocal regulation between14-3-3ζ and MATα1.(8)Co-transfection of 14-3-3ζ and MATα1 or MATα1phosphorylation site mutants examined the liver cancer cell invasion and migration.(9)The effect of the co-expression of 14-3-3ζ and MATα1 or MATα1 phosphorylation site mutants on the growth and metastasis of liver cancer was investigated using a syngeneic metastatic HCC model.Results:(1)Co-IP assay showed that despite lower levels of MATα1 in the cancer tissues,interaction with 14-3-3ζ is higher,and interaction between14-3-3ζ and MATα1 at S180 and T202 site.(2)Interaction between MATα1 and 14-3-3ζ occurs directly and is enhanced by phosphorylation of MATα1 at S180 and T202 by AKT2.(3)When 14-3-3ζ and MATα1 were overexpressed,there is a dramatic reduction in nuclear MATα1 content compared to MATα1overexpression;The double mutant of MATα1 had the most nuclear MATα1 when co-overexpressed with 14-3-3ζ as compared to 14-3-3ζ and MATα1 overexpression.(4)BV02,a 14-3-3 small molecule inhibitor,inhibits the proliferation,invasion and metastasis of liver cancer by blocking the interaction between MATα1 and 14-3-3ζ;BV02 treatment inhibits liver cancer cell growth using a syngeneic model.(5)Overexpressing 14-3-3ζ lowered MATα1 protein and m RNA levels,whereas knockdown of 14-3-3ζ increases MATα1 protein and m RNA levels.Overexpressing MATα1 lowered 14-3-3ζ protein and m RNA levels,whereas knockdown of MATα1 increases 14-3-3ζ protein and m RNA levels.(6)MATα1 acts as a transcriptional repressor cofactor interacting with MAFG or c-MYC,inhibiting their binding to the MARE and E-box elements of the YWHAZ promoter and repressing YWHAZ transcription.(7)14-3-3ζ interacts with FOXM1,C/EBPβ and c-MYC,promoting their binding to the FOX,E-box,C/EBP repressor elements of the MAT1 A promoter and repressing MAT1 A transcription.(8)Co-expression of 14-3-3ζ and MATα1 reversed the inhibitory effect of MATα1 on the migration and invasion of liver cancer;Coexpression of 14-3-3ζ and MATα1 phosphorylation site mutants abolished the migratory and invasive capabilities of 14-3-3ζoverexpression in liver cancer;14-3-3ζ overexpression promoted liver tumor growth and metastasis,whereas MATα1 and MATα1phosphorylation site mutation overexpression inhibited liver tumor growth and metastasis in vivo.Conclusion:(1)14-3-3ζ interacts with MATα1 and segregated MATα1 in the cytoplasm.The S180 and T202 site of MATα1 are responsible for the interaction with 14-3-3ζ.(2)AKT2 is able to phosphorylate MATα1 and contribute to interaction between 14-3-3ζ and MATα1.(3)14-3-3ζ acts as a transcriptional cofactor interacting with FOXM1,C/EBPβ and c-MYC to down-regulate MATα1 expression promoting proliferation,invasion and metastasis of liver cancer.(4)MATα1 acts as a transcriptional cofactor interacting with MAFG and c-MYC to down-regulate 14-3-3ζ expression inhibiting proliferation,invasion,and metastasis of liver cancer.