The Mechanism Research Of NDR Phosphorylates YAP S94 And S127 To Regulate Transcription Activity Of TEAD

YAP is an intracellular connexin and transcriptional coactivator,which mainly binds to the transcription factors TEAD,promotes or enhances the expression of targeting genes.It plays an important role in organ growth,stem cell turnover and cell differentiation.The Hippo pathway is a tumor suppressor pathway,the core of which is MST1/2 and LATS1/2 kinase.The NDR kinase is a LATS1/2 homologue with tumor suppressor function.The Hippo kinase cascade can inhibit YAP transcriptional coactivator.Phosphorylation of YAP S127 retains the cytoplasm of the intestinal epithelial YAP,restricts its transcriptional coactivator function,and inhibits the proliferation of human colon cancer cells.The Hippo-YAP pathway is mostly dysregulated in human tumors.The small molecule that disrupts the YAP-TEAD interaction specifically inhibits the YAP-TEAD interaction and has an anti-tumor effect,thereby provids a new research direction for anticancer drugs.Objective:The purpose of this study was to investigate the effects of NDR on the phosphorylation of YAP S94 and the transcriptional activity of TEAD,and to explore its potential mechanism.methods:1.Phosphorylation of purified YAP protein and NDR protein.Phoshorylation sites of YAP by NDR were measured using mass spectrometry sequencing.Transfected with YAP 5SA in SW480 cells in order to detect the other phosphorylation sites of YAP by NDR by Co-immunoprecipitation test.To detect the activity of TEAD by using luciferase reporter gene assay method.To detect phosphorylation of YAP and TEAD activity by NDR of cells which has transfected with YAP S94A,YAP-NDR plasmid respectively by luciferase reporter gene assay and MTT test.2.To detect the activity of TEAD in cells which has transfected with YAP S94 point mutation(YAP S94A,YAP S94D and YAP S94E),YAP1 WT,YAP-5SA,YAP-5SA/S94A,YAP-5SA/S94D,YAP-5SA/S94E,YAP WT and YAP S94D/S127D plasmid respectively.3.To examine the role of NDR2 on the interaction of TEAD/YAP/14-3-3 and TEAD/YAP S94A by co-Immunoprecipitation assay.Using Western blot,RT-PCR and ubiquitination test to detect TEAD expression levels and ubiquitination on cells which were transfected with up-regultaion of NDR2 plasmid.Results:1.Mass spectrometry analysis showed that there were 9 phosphorylation sites in NDR phosphorylated YAP,the positioning possibilities of YAP S94 and YAP S127 were 100%and 24%,respectively.We use phosphorylated antibody to detect YAP-5SA(5 reported phosphorylation sites:S61,S109,S127,S164,S397 mutated to A).We found that NDR2 still phosphorylates YAP;whereas,when S94 was mutated to A further(YAP-5SA-S94A),the phosphorylation of YAP by NDR2 disappeared.2.TEAD activity was enhanced when cells were transfected with wt-YAP and YAP-5SA plasmid respectively,whereas,TEAD activity was weakened when cells were transfected with YAP S94A and YAP-5SA-S94A respectively.Transfection of YAP WT-NDR,YAP S94A and NDR or YAP S94A/YAP 5SA and NDR decreased the transcriptional activity of TEAD and decreased the cell proliferation ability.Transfection of YAP-5SA/S94A?YAP-5SA/S94D enhanced TEAD transcriptional activity.The TEAD activity wasn't increased obviously when cells were transfected with YAP-5SA/S94A andYAP-5SA/S94E.TEAD activity is more powerful when cells were transfected with YAP-5SA than YAP WT.Transfection of YAP S94D/S127D competitively inhibits the proliferation of YAP WT-activated cells.3.Under the action of NDR2,YAP forms a complex with TEAD and 14-3-3,and the binding ability is enhanced.After up-regulating NDR2,there was no significant change in TEAD mRNA,whereas,ubiquitination hydrolysis of TEAD was enhanced,and TEAD protein levels were reduced.Conclusion:1.In addition to the reported YAP S127 locus,NDR can phosphorylate YAP S94 site.2.Phosphorylation of YAP S94 regulates the binding of YAP to TEAD and the transcriptional activity of TEAD.3.NDR2 induces complex of YAP with TEAD and 14-3-3 and TEAD ubiquitination hydrolysis by phosphorylating YAP S94 and S127.4.YAP S94D can simulate the real phosphorylation state of YAP.5.YAP 5SA-S94A reduced the transcriptional activity of TEAD and attenuated the proliferation of SW480 cells.YAP 5SA-S94D enhances the transcriptional activity of TEAD and enhances the proliferation of SW480 cells.