Fig 2: PPM1B-mediated dephosphorylation of DYRK1A reduces tau phosphorylation at T212.A, HEK293 cells were transfected for 24 h with plasmid encoding V5-Tau, Myc-PPM1A, or Myc-PPM1B alone or in combination. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-V5 antibody. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5 or anti-Myc antibodies. Hsp90 served as a loading control. B, HEK293 cells were transfected for 24 h with plasmid encoding V5-Tau, Xpress-DYRK1A, Myc-PPM1A, or Myc-PPM1B alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5, anti-Xpress, or anti-Myc antibodies. C, HEK293 cells were transfected for 24 h with plasmid V5-Tau, HA-DYRK1A-WT, HA-DYRK1A-S310A, HA-DYRK1A-4SA, or HA-DYRK1A-5SA alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5 or anti-HA antibodies. D, All graph data represent the mean ± SD of three independent experiments (∗∗∗p < 0.001; n.s., not significant). E, HEK293 cells were transfected for 24 h with plasmid encoding V5-Tau, HA-DYRK1A-WT, HA-DYRK1A-S258A, or HA-DYRK1A-S258D alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5 or anti-HA antibodies. The pThr212-tau/tau ratio is shown in (F). All graph data represent the mean ± SD of three independent experiments (∗∗∗p < 0.001). G, HEK293 cells were transfected for 24 h with plasmid encoding V5-Tau, HA-DYRK1A WT, HA-DYRK1A S258A, or Myc-PPM1B alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5, anti-HA, or anti-Myc antibodies. H, all graph data represent the mean ± SD of three independent experiments (∗∗∗p < 0.001; n.s., not significant). I, HEK293 cells were transfected for 24 h with plasmid encoding HA-DYRK1A-WT, HA-DYRK1A-S258A, or HA-DYRK1A-S258D. Cell lysates were immunoprecipitated with anti-HA antibody, and the samples were incubated for 30 min with kinase buffer, recombinant tau, and [γ-32p]ATP. The reaction products were separated by SDS-PAGE and analyzed by autoradiography. Bacterial recombinant tau was stained with CBB. Proper expression of transiently expressed HA-tagged Dyrk1A-WT and its mutants was confirmed by immunoblotting with the anti-HA antibody. The 32P-tau/tau ratio is shown in (J). All graph data represent the mean ± SD of three independent experiments (∗∗p < 0.01; ∗∗∗p < 0.001).

Fig 3: PPM1B has no effect on phospho-DYRK1A at residues S310 or S529.A and B, HEK293 cells were transfected for 24 h with plasmids encoding HA-DYRK1A-S529A and/or Myc-PPM1B. Cell lysates were immunoprecipitated with anti-p-Ser antibody, followed by immunoblotting with anti-HA antibody. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-HA or anti-Myc antibodies. Hsp90 served as a loading control. The ratio of pSer-DYRK1A to DYRK1A is shown in (B). All graph data represent the mean ± SD of three independent experiments (∗∗∗p < 0.001). C, HEK293 cells were transfected for 24 h with plasmids encoding FLAG-DYRK1A-WT, FLAG-DYRK1A-S529A, HA-14-3-3β, or Myc-PPM1B alone or in combination. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-HA antibody. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-Flag, anti-HA, or anti-Myc antibodies. Hsp90 served as a loading control. D, HEK293 cells were transfected for 24 h with plasmids encoding HA-DYRK1A-S529A, HA-DYRK1A-S310/529A, or Myc-PPM1B alone or in combination. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-phospho-serine antibody. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-HA or anti-Myc antibodies. E, all graph data represent the mean ± SD of three independent experiments (∗p < 0.05; ∗∗p < 0.01). F, HEK293 cells were transfected for 24 h with plasmids encoding V5-Tau or HA-DYRK1A-WT, or HA-DYRK1A-S310A, HA-DYRK1A-S529A, HA-DYRK1A-S310/529A, or HA-DYRK1A-K188R (kinase-inactive mutant of DYRK1A) alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5 or anti-HA antibodies. Hsp90 served as a loading control. G, all graph data represent the mean ± SD of three independent experiments (∗∗∗p < 0.001; n.s., not significant).

Fig 4: PPM1B reduces serum-deprivation-induced cytotoxicity in H19-7 cell stably overexpressing DYRK1A through inhibition of toxic tau aggregate formation.A, H19-7 cells stably overexpressing DYRK1A (H19-7/DYRK1A) or control H19-7/pTK cells were incubated in N2 medium for 0 to 72 h, as indicated. Cytotoxicity was measured by assay of LDH release into the supernatant. The graph represents the mean of six independent experiments ± SD (∗∗∗p < 0.001). B‒D, the H19-7/DYRK1A and H19-7/pTK cells were either mock-transfected or transfected for 24 h with plasmid encoding Myc-PPM1B and then incubated in fresh N2 medium for 48 h. B, cytotoxicity was measured by LDH release into supernatant. The graph represents the mean of 12 independent experiments ±SD (∗∗∗p < 0.001). C, cell lysates were analyzed by western blotting with the indicated antibodies. The proper expression of stably and transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-DYRK1A, anti-tau, or anti-Myc antibodies, respectively. Tubulin served as a loading control. D, cells were washed with PBS, fixed with 3.7% formaldehyde, permeabilized with 0.2% Triton-X-100 in PBS, blocked with 1% BSA in PBS, and then stained with anti-Tau (green) and anti-Myc antibody (red). Nuclei were counterstained with DAPI (blue). Representative confocal images of immunostained cells are shown with the low and high magnification. The bottom panels showed enlarged views of the white-boxed area in the upper panels. Scale bar: 20 μm (the upper panel) and 10 μm (the lower panel). The arrowheads indicate tau aggregation dots. E, the percentage of tau-inclusion-positive cells was calculated by dividing the number of cells showing one or more intracellular tau inclusions by total number of tau-stained cells. Tau-inclusion-positive cells were chosen as the cells containing aggregated tau-foci, and the percentage of cells with foci was determined by visual inspection of at randomly chosen fixed areas (n = 9), which have approximately 5 to 10 cells. The three different symbols in the graph (i.e., filled circles, squares, and triangles) represented the data obtained from three independent experiments, respectively. The graph values are expressed as mean ± SD and represents at least three independent experiments (∗∗∗p < 0.001).

Fig 5: DYRK1A binds to PPM1B in mammalian cells.A, HEK 293 cells overexpressing either FLAG-tagged DYRK1A (Flag-DYRK1A) or parental vector (Vector) were immunoprecipitated with anti-FLAG antibody, separated by SDS-PAGE, and the resolved proteins were visualized by Coomassie Brilliant Blue (CBB) G250 staining. As a negative control, cell lysates were immunoprecipitated with preimmune IgG (beads). The four indicated bands (P1‒P4) were then in-gel digested and analyzed by LC-MS/MS. The proper expression of stably transfected proteins in cell lysates was identified with immunoblot analysis using anti-Flag antibody. Hsp90 served as a loading control. B, SH-SY5Y cells were transfected for 24 h with plasmid encoding Xpress-DYRK1A, Myc-PPM1A, or Myc-PPM1B alone or in combination. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Xpress antibody. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-Xpress or anti-Myc antibodies. Hsp90 served as a loading control. C and D, HEK293 cells lysates were immunoprecipitated with anti-PPM1B (C) or anti-DYRK1A antibody (D), followed by immunoblotting with anti-DYRK1A (C) or anti-PPM1B antibody (D). As a control, cell lysates were immunoprecipitated with preimmune IgG (IgG). Asterisk indicates IgG heavy chain. Hsp90 (C) or tubulin (D) served as a loading control, respectively. E, representative confocal images of immunostaining of HEK293 cells expressing both HA-DYRK1A (green) and Myc-PPM1B (red) are shown. Nuclei were counterstained with DAPI (blue). Scale bar = 10 μm.