We recently reported a novel mechanism of anti-apoptotic activity of Bcl-2 by demonstrating a link between an altered mitochondrial metabolism and apoptosis resistance in cells overexpressing Bcl-21. This increase in mitochondrial metabolism was linked to induction of complex IV activity (Cytochrome C oxidase) as well as oxygen consumption2. The pro-oxidant activity of Bcl-2 was a function of an interaction with the small GTPase Rac1. This interaction was validated in a small number of clinical tissues derived from patients with aggressive B cell lymphomas. Interestingly, interrupting the interaction between Bcl-2 and Rac1 restored apoptosis sensitivity of Bcl-2 overexpressing cell lines3. In the present study we set out to identify the domains within the two proteins responsible for the interaction. We show that the pro-oxidant activity of Bcl-2 and its interaction with Rac1 are dependent on the phosphorylation of Bcl-2 at serine 70 within the non-structured loop region adjacent to the BH3 domain. Also, the activation status of Rac1 appears important in this interaction as GDP loading almost completely inhibits, whereas GTP loading facilitates interaction. To further substantiate those findings, isothermal titration calorimetry (ITC) was carried out using Rac1-GTPγS and Bcl-2 BH3 peptides to gain insight into the energetics of binding of BH3 peptide to Rac1-GTPγS. Indeed, the thermodynamic parameters ΔH and ΔS in the ITC experiments of Rac1- GTPγS and BH3 peptide were -2.55 × 104 Joules/mol and +35.6 joules/mol/deg, respectively, and the ΔG values were negative, indicating that the binding of Rac1-GTPγS with BH3 peptide was spontaneous and exothermic and that hydrophobic interactions played a major role; however, hydrogen bonds could not be discounted due to the positive ΔS value and the presence of polar amino acids in the peptide. Similar results were obtained with the S70 phosphorylation mimetic peptide of Bcl-2 (S70E) but not with the S70A phosphorylation dead peptide. Furthermore, the fluorescence intensity of Rac1 decreased steadily with increasing Bcl-2 peptide concentration, indicating an interaction between Rac1 and Bcl-2 peptides. These data provide strong evidence for the existence of a functional interaction between Bcl-2 and Rac1 and for the translational relevance of this interaction in human cancers that are refractory to chemotherapy due to the overexpression of Bcl-2. 1. Chen, Z, X. and Pervaiz, S. Cell Death Diff. 14(9): 1617-27, 2007. 2. Chen ZX, and Pervaiz S. Cell Death Differ. 2010 Mar;17(3):408-20. 3. Velaithan R, et al. Blood. 2011 Jun 9;117(23):6214-26.

Citation Format: Jia Kang, Shani Ajumal, Kunchithapadam Swaminathan, Shazib Pervaiz. Biophysical evidence for the existence of a functional interaction between the small GTPase Rac-1 and the anti-apoptotic protein Bcl-2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 13. doi:10.1158/1538-7445.AM2015-13

©2015 American Association for Cancer Research.
2015