DYRK1A Promotes Cell Migration In Glioblastoma Cells Via Targeting NFATc1

Glioma is the most common brain cancer,and glioblastoma?GBM?is the most common and most aggressive types of glioma.GBM is resistant to chemotherapy and radiotherapy with a median patient survival time of about one year.Since the pathogenesis of glioblastoma is complex and is largely unknown,better understanding of molecular mechanisms of GBM may reveal clues for the design of targeting drugs and development of the new therapeutic strategies.Nuclear factor of activated T cells?NFAT?,originally found in activated T-cells,is a transcription factor that induces gene transcription during immune response.NFATs contain five members:NFAT1?NFATc2,NFATp?,NFAT2?NFATc1,NFATc?,NFAT3?BFATc4?,NFAT4?NFATc3,NFATx?and NFAT5?TonEBP,OREBP?.NFATs were involved in multiple tumor carcinogenesis and NFATcl functions as an oncogenic factor,whereas NFATc2 acts as a tumor suppressor.The activity of NFAT is mediated by phosphorylation and the phosphorylation of NFAT causes its export from the nucleus and loss of transcriptional activity.In our previous study,we demonstrated that DYRK1A phosphorylated NFATc1,thus increasing NFATc1 protein stability,in contrast to decreased NFATc2 protein stability in HEK293T cells.However,whether NFATc1 functions in GBM and the expression of NFATc1 is regulated by DYRK1A is unknown.Here we report that DYRK1A was highly expressed in glioma tumors and glioblastoma cells,and correlating with the high expression of NFATc1.Moreover,inhibition of DYRK1A promoted NFATc1 degradation in GBM cells,and significantly reduced the transactivation of NFATc1.However,DYRK1A showed an opposite effect on NFATc2.Most importantly,our data suggest that DYRK1A inhibition reduced glioblastoma migration.Polypeptide derived from DYRK1A-targeted motif of NFATc1,competitively blocking DYRK1A kinase activity on NFATc1,clearly reduced the expression of NFATc1 and impaired glioblastoma migration.We propose that the high expression of NFATc1 in gliomas promotes glioblastoma migration,which is a key oncogenic event.The polypeptide pharmacological inhibition of DYRK1A could represent a promising therapeutic intervention for GBM.Objective1.Clarify the molecular mechanism of DYRK1A regulation of NFATc12.Clarify the mechanism of DYRK1A regulating the effect of NFATc1 on the occurrence and development of glioblastomaMethods1.The effect of high expression of DYRK1A or knockdown DYRK1A on the expression of NFATc1 and whether it affects the nuclear transfer of NFATc1After the high expression of DYRK1A,the expression level of NFATc1 was increased,while knocking down DYKR1A decreased the expression level of NFATc1.P-nfatc1mycflag was co-transfected into HEK293 cells with PCMV-DyrK1A or PGFP-V-RS-Shdyrkl A,respectively.The cell protein was extracted 48 hours after culture,and the expression of NFATc1 was detected by western blot using mouse anti-Flag antibody.Pcmv-dyrkla was transfected into HEK293 cells,and the difference in expression of endogenous NFATc1 was detected by anti-NFATcl antibody.HEK293 cells were co-transfected with PWT-NFATc2 and PCMV-DyrK1A,and the expression of NFATc2 was detected by anti-HA antibody.1.2 P-NfatCIMyCFlag was co-transfected into HEK293 cells with PCMV-DyrK1A or PGFP-V-RS-ShdyrklA.The cells were collected and then the nuclear and plasmid proteins were isolated.Anti-nfatc1 antibody was used to detect the expression difference of NFATc1 in the nuclear and plasmid.The NFAT activator Ionomycine?ionomycin?was added to HEK293 cells,and the cells were collected to isolate the nuclear and cytoplasmic proteins.Anti-nfatcl antibody was used to detect the difference in the expression of NFATc1 between the nucleus and cytoplasm,and to determine the nuclear transfer of its activated state.2.Verify whether DYRK1A affects the ubiquitination and stability of NFATc1.2.1 HEK293 cells transfected with P-NFATclmycFlag were treated with proteasome inhibitor Laccystin?LAC?,and different action time and concentration were set.HEK293 cells transfected with P-NFATc1mycFlag were treated with lysosomal inhibitor chloroquine?CHL?,and different action time and concentration were also set.Expression of NFATc1 was detected with anti-NFATc1 antibody.2.2 HEK293 cells were transfected with p-NfatCl mycFlag and P-Nfatc2mycFlag high expression plasmids,cullin-1/2DN structure was used as SCF-E3 ligase inhibitor,and anti-NfatC1 and anti-NFATc2 antibodies were used to detect the expression changes of NFATc1 and NFATc2.After the high expression of NFATc1 in HEK293 cells,co-immunoprecipitation?CO-IP?was conducted using anti-Flag antibody and anti-ubiquitin antibodies,and then Western blot?WB?assay was conducted using anti-Ubiquitin and anti-flag antibodies,respectively,to verify the binding of NAFTcl to UBIquitin.3.To verify whether DYEK1A affects the expression of NFATc1 protein by phosphorylation3.1 P-NfatC1Mycflag and PCMV-DyrK1A plasmids were co-transfected into HEK293 cells.Harmine,a DYRK1A inhibitor,was added to the experimental group,and the expression of NFATc1 was detected by anti-flag antibody WB.Pcmv-dyrkla was transfected into HEK293 cells,and the total protein was extracted 48 hours later.The phosphorylated NFATcl and total NFATc1 protein expressions were detected by WB using anti-Pser antibody and anti-NfatCl antibody,respectively.3.2 P-NfatClmyCFlag and PCMV-Dyrk1A plasmids were co-transfected into HEK293 cells,and total proteins were extracted 48 hours later.Co-ip was performed using anti-Flag and anti-dyrklA respectively,and DYRK1A and NFATc1 were detected by WB respectively to verify the interaction between the two.After high expression of DYRK1A,co-IP was performed with anti-Ubiquitin,and the UBI-NFATc1 complex was detected by anti-Flag antibody WB.4.1 Diagram showed NFATs sequence alignment.Sequences of NFATc1?NP₇65978?;NFATc2?NP₇75114?,NFATc3?NP₇75118?,and NFATc4?NP₀01129494?were aligned using ClustalW2 software.Conserved regions among NFATs are underlined.Conserved serine residues in four NFATs are shown in blue.The DYRK1A target motifs are labeled as yellow highlights.The DYRK1A phosphorylation serine residues were in red and bold.4.2 Schematic diagram represents the domain structure of NFATc1 and location of five deletion constructs.AD:activation domain.SPRIEIT:calcineurin binding motif.SP:sp repeats region;SRR:serine-rich region;NLS:nuclear localization sequence.4.3 Five deletion constructs of NFATc1 were co-transfected with pCMV-DYRK1A in HEK293 cells.D.Deletionconstructs of NFATc1 was co-transfected with pCMV-DYRK1A in HEK293 cells.Anti-DYRK1A antibody was used as the immunoprecipitation antibody,and anti-FLAG antibody was used to detect NFATc1 in immunoblotting.5.DYRK1A and NFATc1 protein expression were coordinately increased in brain gliomas.5.1 Representative images for the immunohistochemical staining of DYRK1A and NFATc1 in four different grades of gliomas and normal tissues.DYRK1A was detected with a polyclonal anti-DYRK1A antibody and NFATc1 was detected by a monoclonal antibody in the microarray.DYRK1A protein expression were increased in gliomas.The expression of DYRK1A was quantified by image J software as described in methods.NFATc1 protein levels were increased in gliomas.The expression of NFATcl was quantified by image J software as described in methods.5.2 NFATc1 and DYRK1A were detected by WB in cell lystate of NHA,U87,U251,T98G and HEK293 cell lines.NFATc1 monoclonal antibody and DYRK1A polyclonal antibody were used as described in methods.5.3 Colocalization images for the immunofluorescent staining of DYRK1A and NFATc1 in U251 cells.DYRK1A was detected with a polyclonal anti-DYRK1A antibody and NFATc1 was detected by a monoclonal antibody.6.Transcriptional expression and prognostic significance of DYRK1A and NFATc1.DYRK1A elevated NFATc1 protein expression and transcriptional activity.6.1 The expression of DYRK1A and NFATc1 was compared between T and N in TCGA GBM dataset.C-F.Kaplan-Meier plots were drawn for OS and DFS of patients stratified by tumoral expression of DYRK1A and NFATc1,respectively.P values were obtained by using the log-rank test.Patients were stratified into low and high expression according to median of DYRK1A and NFATc1 mRNA expression.6.2 T98G cells were transfected with pCMV-DYRK1A.NFATc1 and DYRK1A were detected by NFATc1 monoclonal antibody and DYRK1A polyclonal antibody.6.3 T98G cells were co-transfected with NFATc1 activity reporter pNFATluc,p-NFATc1mycflag and pCMV-DYRK1A.Dual luciferase assay was performed 48 hours after transfection.T98G cells were co-transfected with NFATc1 activity reporter pNFATluc,p-NFATc1mycflag and psh-DYRK1A.Dual luciferase assay was performed 48 hours after transfection.T98G cells were cotransfected with p-NFATc1mycflag and pCMV-DYRK1A.qRT-PCR was used to detect the mRNA expression of NFATcl targets IL2 and TNF.6.4 Cytokine array showed differential cytokine expression profiles regulated by DYRK1A in T98G cells.T98G cells was transfected with p-NFATc1mycflag and pCMV-DYRKlA.And a RayBiotech human cytokine antibody array was performed to indicate transcriptional activity of NFATc1 48 hours after transfection.7.DYRK1A decreased NFATc2 protein expression and transcriptional activity.7.1 DYRK1A decreased protein expression of NFATc2 in T98G cells.T98G cells were transfected with pCMV-DYRK1A.NFATc2 and DYRK1A were detected by NFATc2 monoclonal antibody and DYRK1A polyclonal antibody.7.2 The mRNA of NFATc1 target genes IL2 and TNF were decreased by DYRK1A overexpression.pWT-NFATc2 was co-transfected with pCMV-DYRK1A into T98G cells.qRT-PCR was used to detect NFATc2-targeted gene expression of IL2 and TNF.7.3 NFATc2 transcriptional activity was decreased by DYRK1A.T98G cells were co-transfected with NFATc2 activity reporter pNFATluc,p-NFATc2mycflag and pCMV-DYRK1A.Dual luciferase assay was performed 48 hours after transfection.8.DYRK1A regulated NFATc1 to facilitate glioma cell migration.8.1 T98G cells were transfected with DYRK1A and NFATc1 expression vectors alone or simultaneously.Transwell assay showed that co-expression of DYRK1A and NFATc1 greatly increased T98G cell migration.T98G cells were transfected with vectors knocking down DYRK1A and/or NFATc1.Simultaneous knock down of DYRK1A and NFATc1 decreased T98G cell migration.8.2 MTT assay showed T98G cell viability was not changed by expression of DYRK1A and/or NFATc1.D.MTT assay showed T98G cell viability was not changed by knockdown of DYRK1A and/or NFATc1.9.NFATc1-P3 peptide decreased NFATc1 protein level and inhibited T98G cell migration.9.1 Peptide sequences for DYRK1A substrate consensus site and the three phosphorylation sites in NFATc1 protein.The underlined serines indicate the phosphorylation motifs of DYRK1A.T98G cells were transfected by p-NFATclmycflag and pCMV-DYRK1A treated with different concentrations of peptides fused with a HIV-TAT protein for 24 hours after transfection,including HIV-TAT,DYRK1A substrate,NFATc1-P1,NFATc1-P2 and NFATc1-P3.Anti-flag?M2?antibody was used to detect NFATc1 protein and ACTB was used as loading control.9.2 T98G cells were transfected by p-NFATc1mycflag and pCMV-DYRK1A and treated with HIV-TAT?80uM?and NFATc1-P3 fiusion polypeptides?80uM?for 12,24 and 48 hours after transfection.NFATcl protein was detected with anti-flag?M2?antibody and ACTB was used as loading control.9.3 Transwell assay showed that NFATc1-P3 peptide decreased T98G cell migration.T98G cells were cotransfected with p-NFATc1mycflag and pCMV-DYRK1A.24 hours after transfection,cells were treated with HIV-TAT?80uM?and NFATc1-P3 fusion polypeptides?80uM?.Results1.DYRK1A increased NFATc1 protein expression and its nuclear Translocation.1.1 The protein level of NFATc1 was increased with DYRK1A over-expression.And NFATc1 protein expression was decreased with DYRK1A knockdown.1.2 The results showed nucleus and cytoplasm NFATc1 were increased with over-expression DYRK1A,respectively.On the other hand,DYRK1A knock-down decreased cytoplasm and nucleus NFATc1.NFATc1 protein expression was increased in cytoplasm and nucleus by DYRK1A.2.DYRK1A affected NFATc1 protein ubiquitination and stability.2.1 NFATc1 could be degraded by ubi-quitin-proteasome pathway.2.2 Both of NFATc1 and NFATc2 may be degraded through SCF E3 ligase mediated ubiquitin-proteasome pathway.3.DYRK1A affected NFATc1 protein ubiquitination and stability through Phosphorylation.3.1 DYRK1A affected NFATc1 protein phosphorylation.3.2 DYRK1A regulated NFATc1 protein ubiquitination and degradation through phosphorylation.4.Identification of DYRK1A-targeted motifs in NFATc1 protein.4.1 Diagram showed NFATs sequence alignment.Schematic diagram represents the domain structure of NFATc1 and location of five deletion constructs.4.2 DYRK1A had no effects on deleted construct of NFATc1-424-716 that does not containanyputativemotifs.The third and fourth motifs could be the DYRK1A targeting motifs.4.3 S261,S278,S403 and S409 were DYRK1A-targeted motifs.5.DYRK1A and NFATc1 are highly and coordinately expressed in glioma.5.1 The IHC results showed higher expression of DYRK1A in gliomas,and particularly in grade 4 gliomas.5.2 Concomitantly,NFATc1 levels were also significantly higher in gliomas,and especially in grade 4 gliomas,compared with normal tissues.5.3 IHC analysis showed that most samples with positive DYRK1A staining also exhibited strong NFATc1 staining.6.Transcriptional expression of DYRK1A and NFATc1 in glioma,and their association with patient prognosis.DYRK1A increases NFATc1 protein expression and transcriptional activity.6.1 The transcriptional expression of DYRK1A and NFATc1 were both uncorrelated with patient OS and DFS.6.2 NFATc1 was increased by DYRK1A over-expression,and decreased by DYRK1A knockdown.6.3 Over-expression of DYRK1A increased NFATc1 transcriptional activity,while knockdown of DYRK1A decreased NFATc1 transcriptional activity.6.4 Protein panels of various cytokines,including IGFBP4,IL1B,IL6,etc.,were markedly increased by DYRK1A.7.DYRK1A distinctively regulates NFATc1 and NFATc2.7.1 Over-expression of DYRK1A markedly decreased NFATc2 protein level.7.2 Over-expression of DYRK1A decreased the mRNA expression of IL2 and TNF.7.3 DYRK1A knock-down increased the mRNA expression of IL2 and TNF.8.DYRK1A and NFATc1 increase glioma cel migration synergistically.8.1 Co-expression of DYRK1A and NFATc1 greatly increased T98G cell migration,knockdown of both reduced T98G cell migration.8.2 MTT assay showed that the cell viability was not changed by DYRK1A and NFATcl overexpression or knockdown.9.NFATc1 peptides competitively inhibit DYRK1A and glioma cell migration.9.1 NFATc1-P3 significantly inhibited NFATc1 protein level increased by DYRK1A at 80?M concentration.9.2 NFATc1-P3 can inhibit NFATc1 protein expression increased by DYRK1A at 12,24 and 48 hours.9.3 The peptide competitively inhibits the effect of DYRK1A on NFATc1 and glioma cell migration.ConclusionPhosphorylation of NFATc1 by DYRK1A inhibited ubi-quitination and increased NFATc1 protein stability.DYRK1A in the nucleus phosphorylated NFATc1 and made NFATc1 turn out of nucleus.But E3 ligase couldn't link ubiquitin to NFATc1,because of spatial confor-mation of phosphorylated NFATc1 changed,and lead to NFATc1 ubiquitin levels decreased.NFATc1 was not degraded by cytoplasmic proteasome and accumulated in cytoplasm.Inhibition of DYRK1A promoted NFATc1 degradation in GBM cells,and sharply reduced the transactivation of NFATc1,not only decreasing the expression of NFATc1-targeted genes,but also reducing the luciferase activity.Polypeptide pharmacological inhibition of DYRK1A could represent a promising therapeutic intervention for GBM.InnovationThe regulation of NFATc1 by DYRK1A was found to be contrary to the regulation of NFATc2 by DYRK1A as previously reported.DYRK1A increased the protein level and transcription activity of NFATc1,and decreased the transcription activity of NFATc2.Polypeptide pharmacological inhibition of DYRK1A could represent a promising therapeutic intervention for GBM.