DYRK1A phosphorylates MEF2D and decreases its transcriptional activity

Abstract

Myocyte enhancer factor 2D (MEF2D) is predominantly expressed in the nucleus and associated with cell growth, differentiation, survival and apoptosis. Previous studies verified that phosphorylation at different amino acids determined MEF2's transcriptional activity which was essential in regulating downstream target genes expression. What regulates phosphorylation of MEF2D and affects its function has not been fully elucidated. Here, we uncovered that dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A), a kinase critical in Down's syndrome pathogenesis, directly bound to and phosphorylated MEF2D at Ser251 in vitro. Phosphorylation of MEF2D by DYRK1A significantly increased MEF2D protein level but attenuated its transcriptional activity, which resulted in decreased transcriptions of MEF2D target genes. Phosphorylation mutated Ser251A MEF2D exhibited enhanced transcriptional activity compared with wild type MEF2D. MEF2D and DYRK1A were observed co-localized in HEK293 and U87MG cells. Moreover, DYRK1A-mediated MEF2D phosphorylation in vitro might influence its nuclear export upon subcellular fractionation, which partially explained the reduction of MEF2D transcriptional activity by DYRK1A. Our results indicated that DYRK1A might be a regulator of MEF2D transcriptional activity and indirectly get involved in regulation of MEF2D target genes.

Keywords: DYRK1A; MEF2D; glioblastoma; phosphorylation; transcriptional activity.

FIGURE 1

DYRK1A increases MEF2D protein level. (A) HEK293 cells were co‐transfected with MEF2D expression vector together with DYRK1A expression vector or the control vector. Alkaline phosphatase was applied into the cell lysate. Both MEF2D and DYRK1A were detected with anti‐flag antibody. (B) Transfection condition was the same as (A). RNA was isolated 48 h after transfection. Real‐time PCR was performed to examine MEF2D mRNA expression. (C) HEK293 cells were co‐transfected with MEF2D expression vector together with DYRK1A expression vector, DYRK1A kinase dead (DYRK1A‐KD) and the control vector, respectively. MEF2D and DYRK1A were detected by anti‐flag antibody. (D) MEF2D and DYRK1A protein expressions were examined among different glioblastoma cells. MEF2D and DYRK1A were detected with anti‐MEF2D and anti‐DYRK1A antibodies separately. (E) U87MG cells were transfected with DYRK1A expression vector and the control vector. MEF2D and DYRK1A were detected with anti‐MEF2D and anti‐DYRK1A antibodies. (F) Real‐time PCR was performed to examine the mRNA level of MEF2D in (E). (G) Si‐CON and Si‐DYRK1A were transfected into U87MG cells to silence DYRK1A. MEF2D and DYRK1A were detected with anti‐MEF2D and anti‐DYRK1A antibodies. (H) Real‐time PCR was performed to examine the mRNA level of MEF2D in (G). (I) Harmine, a specific inhibitor of DYRK1, was used to treat U87MG cells for 24 h with the concentration at 10 μmol/L. (J) Real‐time PCR was performed to examine the mRNA level of MEF2D in (I). Values represent means ± SD; n = 3; *P < .05 by non‐parametric t test

FIGURE 2

Interaction between DYRK1A and MEF2D. (A) HEK293 cells were transfected with MEF2D expression vector fused with myc and flag tags. Co‐immunoprecipitation (Co‐IP) with anti‐flag antibody was achieved to examine the specific binding of MEF2D and DYRK1A. DYRK1A was detected by anti‐DYRK1A antibody. (B) HEK293 cells were transfected with DYRK1A expression vector fused with myc and flag tags. Anti‐flag antibody was used for IP. MEF2D was detected by anti‐MEF2D antibody. (C) Cell lysates from U87MG cell lysate were immunoprecipitated with anti‐MEF2D antibody. DYRK1A was detected by anti‐DYRK1A antibody. (D) HEK293 cells were co‐transfected with MEF2D‐RFP and pEGFP‐DYRK1A vectors. Confocal microscopy was used to examine the co‐localization of exogenous DYRK1A and MEF2D in HEK293 cells. Co‐localization analysis was performed by ImageJ. (E) Confocal imaging was performed in U87MG cells. Anti‐MEF2D and anti‐DYRK1A antibodies were used to detect endogenous MEF2D and DYRK1A. Co‐localization analysis was performed by ImageJ

FIGURE 3

Mass spectrometric analysis of phosphorylation of MEF2D by DYRK1A in vitro. (A) Purified recombinant human MEF2D and DYRK1A proteins were used for in vitro kinase assay. SDS‐PAGE was performed to obtain MEF2D protein after in vitro kinase assay. (B) Basepeak result of MS for MEF2D without DYRK1A. (C) Basepeak result of MS for MEF2D with DYRK1A. (D) Mass spectrometric analysis of MEF2D phosphorylation. Matching data of 5 fragments indicate the same phosphorylation modification at Serine 251, which was highlighted with arrow

FIGURE 4

Inhibition of transcriptional activity of MEF2D by DYRK1A. (A) U87MG cells were transfected with DYRK1A expression vector or the control vector. RNA was isolated 48 h after transfection. Real‐time PCR was performed to examine mRNA expression of HDAC9 and ZEB1. (B) U87MG cells were transfected with DYRK1B expression vector or the control vector. mRNA levels of HDAC9 and ZEB1 were detected by real‐time PCR. (C) Luciferase assay was performed to examine the transcriptional activity of MEF2D. (D) HDAC9 mRNA level was detected in U87MG cells upon MEF2D variants. (E) ZEB1 mRNA level was detected in U87MG cells. Values represent means ± SD; n = 3; *P < .05 by non‐parametric t test or two‐way ANOVA analysis

FIGURE 5

DYRK1A alters subcellular localization of MEF2D without affecting protein stability. (A) Subcellular fractionation was performed on HEK293 cells that were co‐transfected with MEF2D expression vector together with DYRK1A or the control vector. Anti‐MEF2D and anti‐DYRK1A antibodies were applied. (B) Subcellular fractionation was performed on HEK293 cells that were co‐transfected with MEF2DS251A vector with DYRK1A or the control vector. Anti‐MEF2D and anti‐DYRK1A antibodies were applied. (C) CHX pulse‐chase assay was performed to HEK293 cells that were transfected with MEF2D, MEF2DS251A and MEF2DS251D vectors. Final concentration of CHX is 150 μg/mL. (D) Comparison of wild type MEF2D to MEF2DS251A and MEF2DS251D in CHX pulse‐chase assay. Values represent means ± SD; n = 3; *P < .05 by two‐way ANOVA analysis