Fig. 10
From: Alzheimer risk gene product Pyk2 suppresses tau phosphorylation and phenotypic effects of tauopathy
Pyk2 inhibits LKB1 and p38 MAPK activity. A–C, iPSC-derived human cortical neurons (90–100 days post terminal differentiation) (same as shown in Fig. 2E–I) were treated with PF-719 at indicated concentrations for 2 h at 37 °C and, immediately following treatment, homogenized in RIPA containing 1% SDS. Lysates were separated by SDS-PAGE and immunoblotted with the LKB1 and p38 MAPK antibodies indicated. A, Representative immunoblot images of lysates from PF-719-treated iPSC-derived human cortical neurons. B and C, Quantification of A. Pyk2 inhibition significantly increased LKB1 activity (pLKB1 S428 normalized to total LKB1) at 2.0 μM PF-719 (B) and significantly increased p38 MAPK activity (pp38 MAPK T180/Y182 normalized to total p38 MAPK) at every concentration of PF-719 (C). Data are graphed as mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, n = 6. D–F, TBS-soluble lysates from hippocampi of 9.5–10.5-month-old WT, Pyk2−/−, PS190/+ and PS190/+;Pyk2−/− animals were separated by SDS-PAGE and immunoblotted with the LKB1 and p38 MAPK antibodies listed. D, Representative immunoblot images of TBS-soluble hippocampal lysates. E and F, Quantification of D. A significant increase in TBS-soluble LKB1 activity (pLKB1 S428 normalized to total LKB1) is observed in PS190/+;Pyk2−/− animals compared to WT and Pyk2−/− animals (E), while there were no significant differences in TBS-soluble MAPK activity (pp38 MAPK T180/Y182 normalized to total p38 MAPK) observed across genotypes (F). Data are graphed as mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, n = 7–11 mice. G–K, TBS-insoluble, SDS-soluble lysates from hippocampi of 9.5–10.5-month-old WT, Pyk2−/−, PS190/+ and PS190/+;Pyk2−/− animals were separated by SDS-PAGE and immunoblotted with the antibodies indicated. G, Representative immunoblot images of TBS-insoluble, SDS-soluble hippocampal lysates. Arrowhead indicates pp38 MAPK T180/Y182. H–K, Quantification of G. While no significant differences in TBS-insoluble, SDS-soluble LKB1 activity (pLKB1 S428 normalized to total LKB1) were observed across genotypes (H), PS190/+;Pyk2−/− animals demonstrated a significant increase in TBS-insoluble, SDS-soluble p38 MAPK activity (pp38 MAPK T180/Y182 normalized to total p38 MAPK) compared to WT and Pyk2−/− animals (I). No differences were observed in absolute levels of TBS-insoluble, SDS-soluble phospho-LKB1 (pLKB1 S428 normalized to β-Actin) across genotypes (J), however PS190/+;Pyk2−/− exhibited a significant increase in absolute levels of TBS-insoluble, SDS-soluble phosho-p38 MAPK (pp38 MAPK T180/Y182 normalized to β-Actin) compared to WT, Pyk2−/− and PS190/+ animals (K). Data are graphed as mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ****p < 0.0001, n = 8–16 mice