Figure 8. D319N mutation of ERK2-GFP inhibits GnRH-induced nuclear localization that is not attributable to increases in TEY phosphorylation.

(A) HeLa cells transfected with ERK siRNAs were transduced with Ad wild-type (WT) or D319N-mutated ERK2-GFP as indicated and stimulated with vehicle (Ctrl), 1 nM or 100 nM GnRH for 5 minutes prior to staining, imaging and analysis as described in Methods. The plots show comparison of average ERK2-GFP N:C ratio in cell populations within defined bins of ppERK2-GFP staining intensity (80 AFU per bin, accepting a minimum of 50 cells per bin in each experiment). Data shown are mean ±SEM (n = 3). Two way ANOVAs revealed ppERK2-GFP as a significant source of variation for control cells and for cells receiving either GnRH concentration. The D319N mutation was a significant variable (P<0.05) only for cells receiving 100 nM GnRH and in this case, post-hoc Bonferroni tests revealed significant differences between WT- and D319N-ERK2 expressing cells in the lowest 2 ppERK2 bins (P<0.05). Only paired data (i.e. the three bins where data are available with and without inhibitor) were used in this analysis. (B) The graphs show full concentration-response curves of ERK2-GFP N:C ratio (right panel) for cells treated as in panel A, using only cells within a defined range (160–240 AFU) of ppERK2-GFP staining intensity present at all levels of stimulus (left panel). Data are mean ± SEM (n = 8). Two way ANOVA for the ERK2-GFP N:C data revealed both GnRH concentration and D319N mutation as significant variables (P<0.01) and post-hoc Bonferroni tests revealed significant differences between WT- and D319N-ERK2 expressing cells receiving 10 or 100 nM GnRH (*, P<0.05).