Abstract 21166: TNF-alpha-mediated MAPK-activated Protein Kinase 2-SUMOylation Inhibits MK2 Kinase Activation, HSP27 Phosphorylation, and Subsequent Endothelial Migration: Involvement of MK2-Mediated Stress Fiber Formation and Actin Remodeling
Stress fiber formation (SFF) regulates a number of endothelial cell (EC) processes like migration and alignment. We found that both tumor necrosis factor (TNF) and steady laminar shear stress (s-flow) increased SFF, but only TNF inhibited EC migration through excessive SFF. It is well known that MAPK-activated protein kinase (MK2)-mediated phosphorylation of heat shock protein-27 (HSP27) promotes SFF, but the role of this mechanism in TNF and s-flow signaling remains unclear. We found that TNF induced MK2-SUMOylation at the lysine-339 site in EC. Therefore, we hypothesized that MK2-SUMOylation contributes to TNF-mediated SFF and the inhibition of EC migration. Loss of the K339 MK2-SUMO site (MK2-K339R) enhanced MK2 kinase activity versus wild-type MK2 (WT-MK2) and prolonged TNF-mediated HSP27 phosphorylation in EC using MK2 adenoviral constructs (fold increase WT 2.21±0.539 vs. K339R 7.942±0.229, p < 0.01). TNF significantly increased SFF in EC, and dominant-negative MK2 (DN-MK2) significantly inhibited SFF. In contrast, SFF was increased by WT-MK2 and more robustly with the MK2-K339R mutant, suggesting that TNF-mediated MK2-SUMOylation at the K339 site functions as a negative regulator of HSP27 phosphorylation and subsequent SFF. Next, we determined the role of MK2 on s-flow-mediated actin remodeling. Six hour EC alignment under s-flow (24 dynes/cm2) was enhanced by WT-MK2 and significantly more by MK2-K339R, but completely inhibited by DN-MK2 (% alignment WT 48.15±5.30, DN 14.89±3.29, K339R 95.21±1.522, p < 0.01). These data suggest the role of MK2 on both TNF and s-flow-mediated SFF, and the inhibitory effect of MK2-SUMOylation on actin remodeling. Lastly, we determined the role of MK2 on EC migration by wound healing assay, and found that MK2-K339R significantly accelerated TNF-mediated inhibition of EC migration compared with WT-MK2. In conclusion, our data suggests the role of TNF-mediated MK2-SUMOylation as a negative regulator of MK2-mediated actin remodeling to prevent excessive SFF and inhibition of vascular repair.
- © 2010 by American Heart Association, Inc.