The Bcl-2/Bcl-xL inhibitor BH3I-2a2 affects the dynamics and subcellular localization of sumoylated proteins

Sumoylation modulates many proteins implicated in apoptosis such as Fas, TNFR1, Daxx, p53 and its regulator MDM2. Some of these proteins, such as DRP-1, are involved in the intrinsic apoptosis pathway. The intrinsic pathway is regulated at the mitochondrial level by the Bcl-2 family of proteins. The small-molecule inhibitor BH3I-2a2 binds to the hydrophobic groove of the BH3 domain of anti-apoptotic proteins Bcl-xL and Bcl-2. Following treatment with this inhibitor in various experimental conditions, we observed decreased levels of detergent-soluble SUMO-1, an increase in the relative levels of detergent-insoluble sumoylated proteins, or both. Accordingly, immunofluorescence microscopy revealed that the relative numbers and intensities of endogenously or exogenously expressed SUMO-1 foci in the nucleus were increased following BH3I-2a2 treatment. MG132 caused a large increase in steady-state levels of SUMO-1 and of sumoylated proteins, and this was especially true for detergent-insoluble proteins. The conjugation-incompetent GG-to-AA SUMO-1 mutant, which did not form nuclear foci, was only present in the detergent-soluble lysate fraction and was insensitive to BH3I-2a2, implying that BH3I-2a2 specifically affects SUMO in its conjugated form. Finally, BH3I-2a2 had similar effects on SUMO-2 and SUMO-3 as it had on SUMO-1. In conclusion, BH3I-2a2 causes an intracellular redistribution of sumoylated proteins, more specifically their targeting to PML and non-PML nuclear bodies in which they may be degraded by the proteasome. Interestingly, knocking down Bcl-2 also altered levels of sumoylated proteins and their presence in detergent-insoluble compartments, confirming the role of Bcl-2 as a modulator of the sumoylation pathway.