Quiescent endothelial cells contain low concentrations of plasminogen activator 
inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a 
variety of inflammatory mediators. In this study, we provide evidence that PAI-2 
interacts with proteasome and affects its activity in endothelial cells. To 
ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were 
coimmunoprecipitated from endothelial cells and identified with specific 
antibodies. The specificity of this interaction was evidenced after (a) 
transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both 
proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using 
specific small interfering RNA (siRNA). Subsequently, cellular distribution of 
the PAI-2·proteasome complexes was established by immunogold staining and 
electron microscopy analyses. As judged by confocal microscopy, both proteins 
appeared in a diffuse cytosolic pattern, but they also could be found in a dense 
perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting 
that it bound to proteasome not as the substrate but rather as its inhibitor. 
Consistently, increased PAI-2 expression (a) abrogated degradation of degron 
analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) 
prevented degradation of p53, as evidenced both by confocal microscopy and 
Western immunoblotting, and (c) inhibited proteasome cleavage of specific 
fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced 
with inflammatory stimuli, can inhibit proteasome and thus tilt the balance 
favoring proapoptotic signaling.