FIGURE 3.
Expression of the human kinases GRK5/PLK2 increases αSyn Ser-129 phosphorylation in yeast. A, Smt3ts mutant cells co-expressing αSyn and GRK5 or PLK2 at permissive (25 °C; +SUMO) or restrictive temperature (30 °C; −SUMO). The phosphorylation level of αSyn on Ser-129 was detected by αSyn Ser-129 phosphorylation-specific antibody (αSyn pSer129) when expressed either alone (αSyn-GFP + empty vector (EV)) or in the presence of GRK5 or PLK2. Immunoblotting analysis of yeast cells expressing S129A-GFP with αSyn Ser(P)129 antibody (right panel) was used as a control for antibody specificity. B, quantification of αSyn Ser-129 phosphorylation level in the presence or absence of GRK5 and PLK2, respectively, at permissive (25 °C; +SUMO) or restrictive temperature (30 °C; −SUMO). Densitometric analysis of the immunodetection of αSyn Ser(P)-129 was normalized to the total amount of αSyn and relative to αSyn + EV at permissive temperature (25 °C; +SUMO). Significance of differences was calculated with one-way ANOVA with Bonferroni's multiple comparison test (**, p < 0.01; ##, p < 0,05 versus empty vector, n = 4). C, Western hybridization of W303 yeast cells co-expressing K96R/K102R-GFP and GRK5 or PLK2. The phosphorylation level of sumoylation-deficient αSyn mutant on Ser-129 was visualized with αSyn Ser(P)-129. D, quantification of αSyn Ser-129 phosphorylation levels of sumoylation-deficient αSyn mutant in the presence or absence of GRK5 and PLK2. Densitometric analysis of the immunodetection of αSyn Ser(P)-129 was normalized to the total amount of αSyn. Significance of differences was calculated with one-way ANOVA (**, p < 0.01, n = 4).