Abstract 422: Activation of the p38 Branch of Mitogen Activated Protein Kinase Pathway Stimulates Proteasome Proteolytic Function

Ubiquitin-proteasome system (UPS) dysfunction is associated with development of most heart diseases. Proteasomal functional insufficiency has been experimentally demonstrated to play an important role in cardiac pathogenesis. Hence, enhancing proteasome function is potentially a powerful new strategy for therapeutic exploration but this research area is hindered critically by poor understanding of the regulation of cellular proteasomes. Here, we sought to determine whether and how the p38 branch of mitogen activated protein kinase (MAPK) regulates the UPS. GFPu is a rationally modified green fluorescence protein (GFP) proven to be a UPS-specific substrate. In both an HEK293-GFPu/RFP stable cell line and cultured neonatal rat ventricular myocytes (NRVMs), serum withdrawal activated p38 and decreased GFPu/RFP protein ratios. The decrease was abolished by p38 inhibitor (SB230580). Similar effects were also observed when p38 was activated by anisomycin. Cycloheximide (CHX) chase assays showed that p38 activation by either anisomycin treatment or MKK3 overexpression significantly shortened the half-life of GFPu proteins. These compelling data demonstrate for the first time that p38 stimulates UPS performance. To pinpoint the step of the UPS pathway which p38 acts on, we examined the level of total ubiquitin conjugates and proteasomal peptidase activity. Activation of p38 via various means failed to increase the level of total ubiquitin conjugates but was able to increase significantly proteasomal chymotrypsin-like activity in cultured NRVMs and this increase was remarkably blunted by SB230580. The altered proteasome activity was not associated with discernible changes in the protein abundance of representative subunits of the 19S (Rpt6, Rpn2) and 20S proteasomes (α5, β5, β7); however, 2-dimensional PAGE followed by immunoblot showed that the isoelectric point (pI) of a fraction of β5, but not α2, subunits of the 20S proteasome was shifted to the acidic side, consistent with increased phosphorylation, by MKK3 overexpression in cultured NRVMs. It is thus demonstrated for the first time that p38 MAPK stimulates proteasome proteolytic function and it achieves such effect likely through post-translational modifications of the proteasome.