Phosphorylation and inactivation of glycogen synthase kinase 3β (GSK3β) by dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A)

Abstract

Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr(356) residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)(356)-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β.

Keywords: Dyrk1A; Enzyme Inactivation; Glycogen Synthase Kinase 3 (GSK-3); Obesity; Phosphorylation; Serine/Threonine Protein Kinase.

FIGURE 1.

Dyrk1A interacts with GSK3β. A and B, co-IP assays in HEK293T cells. HEK293T cell lysates that were transfected with plasmids encoding GSK3β and Dyrk1A were immunoprecipitated with control IgG, anti-GSK3β (A), or anti-Dyrk1A antibodies (B) and then subjected to immunoblot analysis with the indicated antibodies. C, co-IP in brain lysates. Mouse brain lysates were immunoprecipitated with control IgG or anti-Dyrk1A antibodies and then subjected to immunoblot analysis with the indicated antibodies. D, GST pulldown assay to determine the direct interaction between GSK3β and Dyrk1A. Purified GST or GST-GSK3β fusion protein that was immobilized on beads was incubated with recombinant Dyrk1A protein and subjected to immunoblot analyses.

FIGURE 2.

GSK3β is phosphorylated by Dyrk1A at the Thr³⁵⁶ residue in vitro. A, autoradiograph of SDS-polyacrylamide gels that contained the products of in vitro kinase assays with an inactive form of GSK3β (GSK3β K85R) substrate and Dyrk1A WT or the Y321F kinase-inactive mutant. B, top row, autoradiograph of SDS-polyacrylamide gels that contained the products of the in vitro kinase assays that were performed using GSK3β K85R substrates (WT and mutants) and Dyrk1A. Bottom row, Coomassie staining of SDS-polyacrylamide gels that contained purified recombinant WT and mutant GSK3β proteins. C, left panel, autoradiography of SDS-polyacrylamide gels that contained the products of in vitro kinase assays that used GSK3β deletion mutant proteins and Dyrk1A. Right panel, Coomassie staining of SDS-polyacrylamide gels that contained purified GSK3β deletion mutant proteins. D, autoradiography (top panel) of SDS-polyacrylamide gels that contained the products of in vitro kinase assays that employed purified GSK3β (343–420) or GSK3β (343–420) (T356A) mutants in the presence or absence of Dyrk1A. Bottom panel, Coomassie staining of SDS-polyacrylamide gels that contained purified GSK3β mutant proteins.

FIGURE 3.

Dyrk1A-mediated phosphorylation of GSK3β at Thr³⁵⁶ inhibits its activity in vitro. A, purified GST-GSK3β protein that was immobilized on glutathione-Sepharose beads was incubated with Dyrk1A in the presence or absence of ATP. The bead-bound GST fusion proteins were washed and subjected to kinase assays with a GSK3 substrate peptide, GSM. ***, p < 0.001 versus ATP by Student's t test. B, purified GST-GSK3β proteins, either WT or mutant forms (T356A, T356E, or S389A) were subjected to kinase assays with a GSM substrate peptide. Coomassie staining of SDS-polyacrylamide gels that contained purified recombinant WT and mutant GSK3β proteins are shown on the right. **, p < 0.01; ***, p < 0.001 versus GSK3β WT; ###, p < 0.001 versus GSK3β T356A; +++, p < 0.001 versus GSK3β T356E in analysis of variance followed by Bonferroni post hoc test. C and D, autoradiographs (top panels) and Coomassie staining (bottom panels) of SDS-polyacrylamide gels containing the products of in vitro kinase assays that used purified RCAN1 proteins (C) or C-terminal fragment of amyloid precursor protein (APPct, D) in the presence or absence of GSK3β (WT or T356A mutant).

FIGURE 4.

Dyrk1A-mediated phosphorylation of GSK3β at Thr³⁵⁶ inhibits its activity in cells. A, GSK3β, immunoprecipitated using an anti-Myc antibody from lysates of HEK293T cells that were transfected with plasmids that encode Myc-tagged GSK3β WT or mutants were subjected to kinase assays. The immunoblot shown below depicts immunoprecipitated GSK3β WT and mutants. **, p < 0.01; ***, p < 0.001 versus GSK3β WT, ###, p < 0.001 versus GSK3β T356A; +, p < 0.05 versus GSK3β T356E. B, the lysates of HEK293T cells that were transiently transfected with a Tau expression plasmid either in the presence or in the absence of a plasmid encoding GSK3β WT or indicated mutants were subjected to immunoblotting with the indicated antibodies. The results shown in the left panel are representative immunoblots. The densitometric analysis of the immunoblots is shown in the right panel. **, p < 0.01 versus GSK3β WT. C, HEK293T cells were transfected in triplicate with an NFAT-luciferase reporter construct and the indicated amount of GSK3β. Cells were treated with ionomycin for 48 h and analyzed for reporter gene activity. Luciferase activity was plotted as the percentage of the group without GSK3β. **, p < 0.01; ***, p < 0.001 versus GSK3β (0 ng). D, representative experiments measuring NFAT-luciferase transcriptional activity of GSK3β and mutants were performed in quadruplicate. 200 ng of GSK3β WT and the mutants was used for the assay. Luciferase activity was plotted as the percentage of activity compared with the control. ***, p < 0.001 versus control (Con). E, inhibition of luciferase activity by GSK3β mutants is plotted as the percent of activity compared with GSK3β WT. *, p < 0.05; ***, p < 0.001 versus GSK3β WT. All data are represented as mean ± S.E. and were analyzed by analysis of variance followed by Bonferroni post hoc test.

FIGURE 5.

Dyrk1A TG mice are lean and resistant to diet-induced obesity. A and B, body weight of Dyrk1A TG (closed circles) and control male mice (open circles) fed a normal chow diet (A) or 45% HFD (B). Body weight was measured once per week starting at 5 weeks of age (n = 4∼6). C, average weekly food intake per male mouse body weight during weeks 5–18 for mice fed a chow diet or a 45% HFD. D, energy expenditure of Dyrk1A TG mice fed a 45% HFD for 15 weeks from week 5 (n = 5). E and F, epididymal and perirenal (with kidney weight) fat content of 7-month-old control and Dyrk1A TG mice that were fed a chow diet (E) or of mice that were fed a 45% HFD (F) starting at 5 weeks of age and continuing for 10 weeks. The fat weight of each mouse was normalized by its body weight. G, representative MRI images of 3- to 7-month-old control and Dyrk1A TG mice that were fed a chow diet. H, ventral and dorsal views of 7-month-old control and Dyrk1A TG mice that were fed a chow diet. I, reduced adipocyte size of Dyrk1A TG mice. Sections from epididymal fat pads of 7-month-old control and Dyrk1A TG mice were stained with H&E. Scale bars = 126 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control mice by Student's t test.

FIGURE 6.

Specificity of Thr(P)³⁵⁶-GSK3β antibody and the expression of Thr(P)³⁵⁶-GSK3β in WAT and 3T3-L1 cells. A, purified GSK3β proteins were subjected to kinase assays in the presence or absence of Dyrk1A (WT or inactive Y321F mutants). The reaction mixtures were subjected to SDS-PAGE and immunoblotting with phospho-GSK3β antibody. B, HEK293T cells that were transfected with GSK3β WT or GSK3β T356A expression plasmids in the presence or absence of plasmids encoding Dyrk1A were analyzed by immunoblots with the indicated antibodies. C, the lysates of GSK3β WT or GSK3β^−/− mouse embryonic fibroblast cells were analyzed by immunoblot with the indicated antibodies. D, the lysates of WAT were treated with (+) or without (−) λ-protein phosphatase and subsequently analyzed by immunoblot with the Thr(P)³⁵⁶-GSK3β or GSK3β antibodies. E. Peptide competition assay for the pT356-GSK3β antibody. Mouse WAT lysates were analyzed by immunoblot with the Thr(P)³⁵⁶-GSK3β antibody that was preincubated in the absence (N) or presence of GSK3β-non-phosphopeptide (NP) or GSK3β-phosphopeptide (P). F, 3T3-L1 cells transfected with Dyrk1A-specific or control siRNA were analyzed by immunoblot with the indicated antibodies. G, 2-day post-confluent 3T3-L1 preadipocytes (day 0) were induced to differentiate as described under “Experimental Procedures.” Differentiated 3T3-L1 cell lysates were analyzed by immunoblot with the indicated antibodies.

FIGURE 7.

Thr(P)³⁵⁶-GSK3β is increased in the WAT of Dyrk1A TG mice that were fed a normal diet. Shown are representative immunoblots (A) and densitometric analysis (B) of WAT lysates of Dyrk1A TG mice and control (Con) mice fed a normal chow (n = 4∼6). WAT lysates of mice at 5–9 months of age were used for analysis. The phospho-GSK3β signals in the immunoblots were normalized by GAPDH and GSK3β signals. The GSK3β, GS, and β-catenin signals were normalized by GAPDH. *, p < 0.05; **, p < 0.01 versus control mice by Student's t test.

FIGURE 8.

Thr(P)³⁵⁶-GSK3β is increased in the WAT of Dyrk1A TG mice that were fed a HFD. A and B, representative immunoblots (A) and densitometric analysis (B) of WAT lysates of 5–9 month-old Dyrk1A TG and control (Con) mice fed a HFD (n = 6∼10). The amounts of phospho-GSK3β, GS, and β-catenin of Dyrk1A TG are plotted as a percentage compared with control mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control mice by Student's t test. C, body weight of obese and lean control littermates fed an HFD. *, p < 0.05 versus obese mice by Student's t test. D and E, representative immunoblots (D) and densitometric analysis (E) of WAT lysates of obese and lean mice that were fed a HFD (n = 5). WAT lysates of mice at 5–9 months of age were used for analysis. The amounts of phospho-GSK3β, GS, and β-catenin of lean mice are plotted as a percentage compared with obese mice. Data are represented as means ± S.E. *, p < 0.05; **, p < 0.01 versus obese mice by Student's t test.

FIGURE 9.

Effect of Dyrk1A overexpression on preadipocyte differentiation and expression of adipogenesis marker proteins. A, 3T3-L1 cells overexpressing Dyrk1A and control (Con) cells were differentiated for 8 days and then fixed and stained with Oil Red O. B, 3T3-L1 cells overexpressing Dyrk1A and control cells were differentiated for the indicated number of days and analyzed by Western blot to characterize protein expression. C, representative immunoblots of WAT lysates of 8-week-old Dyrk1A TG and control mice fed a normal chow diet.

FIGURE 10.

Schematic for the hypothetical role of Dyrk1A in the mechanism of obesity resistance. ↑, increase; ↓, decrease; −, no change.