Figure 1.
Effect of ERK binding domain and phosphatase activity on MKP-3 promoted transcription of gluconeogenic genes and FOXO1 nuclear translocation.
A. Overexpression of MKP-3 WT or mutants deficient in ERK binding domain, deficient in phosphatase activity or deficient in ERK phosphorylation sites in Fao cells and the effect on phosphorylation of ERK and FOXO1. MKP-3 WT or mutants were over-expressed in Fao cells via adenovirus-mediated gene transfer. B. Effect of MKP-3 wild type (WT) or mutants on transcription of PEPCK and G6Pase promoters in Fao cells. Results were presented as ratios of firefly luciferase activities (FF) versus renilla luciferase activities (RL). C. Effect of MKP-3 WT or mutants on nuclear translocation of FOXO1-GFP in Fao cells. Vec, vector; Luc, luciferase; veh, vehicle; Dex, dexamethasone. *, P<0.05, MKP-3 wild type or mutant expressing cells versus control cells expressing an empty vector (B) or an inactive kinase (C).
Figure 2.
Effect of MKP-3 ERK binding domain and phosphatase activity on expression of endogenous G6Pase gene and glucose output in Fao cells.
A. Over-expression of MKP-3 WT or mutants in Fao cells and the effect on expression of endogenous G6Pase gene. B. Overexpression of MKP-3 WT or mutants in Fao cells and the effect on glucose output. *, P<0.05, MKP-3 wild type or mutant expressing cells versus control cells expressing GFP.
Figure 3.
Mapping the functional domains of MKP-3 essential for mediating the interaction between MKP-3 and FOXO1.
A. Co-immunoprecipitation of FOXO1 WT and MKP-3 WT or mutants deficient in ERK binding domain, deficient in phosphatase activity or deficient in ERK phosphorylation sites from Fao cells. B. Co-immunoprecipitaion of FOXO1 WT and MKP-3 WT or mutants truncated from C terminus.
Figure 4.
Mapping the functional domains of FOXO1 essential for mediating the interaction between MKP-3 and FOXO1.
A. Co-immunoprecipitaion of FOXO1 WT or mutants truncated from C terminus and MKP-3 WT from Fao cells. FOXO1 WT or mutants and MKP-3 WT were co-expressed in Fao cells via adenovirus-mediated gene transfer. GFP was used as a control. B. Co-immunoprecipitaion of FOXO1 WT or mutants deficient in Akt or ERK phosphorylation sites and MKP-3 WT.
Figure 5.
Akt/ERK phosphorylation resisitant FOXO1 mutants and MKP-3 promoted transcription of gluconeogenic genes.
A. Effects of FOXO1 3A and 9A on transcription of PEPCK and G6Pase promoters. *, P<0.05 compared to vector control; #, P<0.05 compared to FOXO1 WT. B. Effects of FOXO1 3A and 9A on transcription of PEPCK and G6Pase promoters in the presence of MKP-3.
Figure 6.
The Akt phosphorylation resistant FOXO1 mutant (FOXO1 3A) rescues the hypoglycemic effect caused by reduced MKP-3 expression in the liver of lean mice.
A. Body weight and glucose levels in mice injected with adenovirus expressing shGFP, shMKP-3, GFP or FOXO1 3A (n = 8 for each froup). B. Protein expression of MKP-3 and FOXO1 in mice as described in A (n = 4 for each group). C. Quantitative graphs for B. D. Expression of gluconeogenic genes in mice as described in A (n = 8 for each group). *, P<0.05, between groups as indicated on graphs.