Figure 9.

ERK8 activates Rho to regulate DAT. A, Cells transfected with HA-ERK8 plasmid show an increase in active RhoA protein compared with mock transfected cells. Active RhoA was measured by pulldown using GST-RBD beads that bind active Rho. B, Quantification of three experiments. Active RhoA was increased in HA-ERK8 transfected cells (t₍₄₎ = 3.05, p < 0.05, unpaired t test), with no significant change in total RhoA protein levels (t₍₄₎ = 0.77, p = 0.49, unpaired t test). Significance was calculated using a two-tailed Student's t test comparing empty vector and ERK8 transfection for both total and active RhoA with significance set at p < 0.05. *p < 0.05. C, As shown previously, ERK8 overexpression significantly increases DAT activity compared with vector-transfected cells (t₍₈₎ = 4.47, p < 0.01, Bonferroni's post-tests). Transfection of GFP-C3 prevents any increase in DAT activity with ERK8 overexpression (DAT/ERK8/GFP-C3 vs DAT/GFP-C3: t₍₈₎ = 0.43, p > 0.05, Bonferroni's post-tests). Data were analyzed using a one-way ANOVA with Bonferroni's post-tests comparing DAT/ERK8 to DAT, and DAT/ERK8/GFP-C3 to DAT/GFP-C3. Significance was set at p < 0.05. **p < 0.01. D, Transgenic expression of Pdat-1::rho-1 cDNA in swip-13(gk1234) animals drives overexpression of WT rho-1 in DA neurons, and significantly rescues Swip compared with nontransgenic swip-13(gk1234) animals (t₍₅₉₎ = 15.85, p < 0.0001, unpaired t test). Bars represent the average of three transgenic lines with at least 100 animals per line. Significance was calculated using a two-tailed Student's t test with significance set at p < 0.05. ****p < 0.0001. E, Treatment of N2 animals with the ROCK inhibitor fasudil HCl induces significant Swip that is significantly reduced in cat-2(e1112) and cat-2(tm2261) mutants. Significance was calculated using a one-way ANOVA with Bonferroni's post-tests comparing N2 in water to N2 in fasudil (t₍₁₅₆₎ = 16.58, p < 0.0001, Bonferroni's post-tests), N2 in fasudil to cat-2(e1112) in fasudil (t₍₁₅₆₎ = 6.41, p <0.0001, Bonferroni's post-tests), and N2 in fasudil to cat-2(tm2261) in fasudil (t₍₁₅₆₎ = 6.19, p <0.0001, Bonferroni's post-tests). Significance was set at p < 0.05. ****p < 0.0001.