Abstract 540: Angiotensin II and Reactive Oxygen Species (ROS) Induce ERK5-SUMOylation and Inhibit ERK5 Transcriptional Activity: Implication in Diabetic Cardiomyopathy (DMC)
BACKGROUND SUMOylation is a novel post translational modification system and affects on protein localization and inhibits transcriptional activities. ERK5 is a member of the MAP kinase family, which has unique carboxyl-terminal domain containing transcriptional activation. Cardiac specific constitutively active form of MEK5α (CA-MEK5α) prevented cardiac dysfunction and apoptosis induced by ischemia/reperfusion. In the present study, we investigated the role of Ang II and ROS in regulating ERK5 kinase and its transcriptional activity in heart.
METHOD AND RESULTS ROS increased both ERK5 phosphorylation and kinase activity. In contrast, H2O2 (100 μM) and Ang II (200 nM) significantly inhibit CA-MEK5α-mediated ERK5 transcriptional activity (8.75 ± 0.5 vs 5.17 ± 0.7, p<0.05). Since Ang II and H2O2 caused significant band shift of ERK5, we investigated whether ERK5 can be modified by SUMOylation. We co-transfected SUMO1 or SUMO2 with Flag-ERK5 in CHO cells, and observed huge ERK5-SUMOylation by co-immunoprecipitation of ERK5 with SUMO2 but not SUMO1. Since a consensus sequence Ψ-K-x-D/E is required for SUMO modification, we created a point mutation at K6R and K22R, and found ERK5-SUMOylation was decreased in double mutant of K6R/K22R, suggesting both K6 and K22 are SUMOylation sites. Next, to determine the role of ERK5-SUMOylation on ERK5 transcriptional activity, we compared CA-MEK5α-mediated ERK5 transcriptional activity between wild type and mutants using Gal4-ERK5 reporter gene transfection. We found that the inhibition of ERK5 transcriptional activity induced by SUMO2 was prevented in K6R/K22R ERK5 mutant, which suggested the critical role of ERK5- SUMOylation in ERK5 transcriptional activity. It is well known that Ang II and H2O2 play a major role in the development of DMC. We investigated ERK5-SUMOylation in diabetic and ischemic heart. We treated mice with streptozotocin (DM group) to induce diabetes. One week after injection, myocardial infarction (MI) was induced. There are concomitant induction of ERK5-SUMOylation in DM + MI group with exacerbation of LV remodeling after MI.
CONCLUSION These results demonstrated that critical role of ERK5-SUMOylation in regulating its transcriptional activity, and its possible role in diabetic heart.