Interaction of Caveolin-1 with Ku70 Inhibits Bax- Mediated Apoptosis Huafei Zou, Daniela Volonte, Ferruccio Galbiati* Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America Abstract Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding domain (CBD) of Ku70 (amino acids 471–478). Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug- induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 WRA), limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death. Citation: Zou H, Volonte D, Galbiati F (2012) Interaction of Caveolin-1 with Ku70 Inhibits Bax-Mediated Apoptosis. PLoS ONE 7(6): e39379. doi:10.1371/ journal.pone.0039379 Editor: Libing Song, Sun Yat-sen University Cancer Center, China Received June 16, 2011; Accepted May 24, 2012; Published June 20, 2012 Copyright: 2012 Zou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institute on Aging (R01-AG022548 and R01-AG030636) (to F.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: feg5@pitt.edu Introduction Caveolae are 50 to 100 nm flask-shaped invaginations of the plasma membrane enriched in cholesterol and glycosphingolipids. Caveolae can exist as individual invaginations or are found in detached grape-like clusters and long tubular structures derived from the fusion of single caveolae within the cytoplasm. Caveolae were identified in the 1950’s in capillary endothelial cells and epithelial cells from the gall bladder of the mouse [1,2]. Since then, caveolae have been found in a wide variety of tissues and cell types, including adipocytes, endothelial cells, striated and smooth muscle cells, fibroblasts and epithelial cells [3]. Caveolae were originally thought to function as macromolecular transport vesicles [4]. From then on, their role has expanded to signal transduction, cellular metabolism, cholesterol homeostasis, endocytosis and tumor suppression [5–9]. Caveolin-1 is an essential protein component of caveolae. Caveolin-1 acts as a scaffolding protein that concentrates and functionally regulates signaling molecules, including G-protein alpha subunits, H-Ras, Nitric Oxide Synthase (NOS), Epidermal Growth Factor Receptor (EGFR), Src-like Nonreceptor Tyrosine Kinases (NRTK), Protein Kinase C (PKC), Protein Kinase A (PKA), Mdm2, Thioredoxin Reductase 1 and Protein Phosphatase type 2A [10–13]. The direct interaction between caveolin-1 and signalling molecules occurs between the ‘‘caveolin scaffolding domain’’ (CSD) of caveolin-1, which is represented by residues 82–101, and the caveolin binding domain (CBD: WXWXXXXW, WXXXXWXXW, or WXWXXXXWXXW where W represents an aromatic amino acid and X represents any amino acid) of signaling molecules [14,15]. The direct interaction with caveolin-1 generally results in the sequestration of a given signaling molecule within caveolar membranes and inhibition of its signaling activity. Colorectal cancer is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related deaths in the United States [16,17]. Apoptosis is an anti-tumorigenic event. In fact, tumor cells need to escape apoptosis in order to generate a relevant tumor mass. Thus, apoptosis has a profound effect on the malignant phenotype. In addition, most cytotoxic anticancer agents promote apoptosis, suggesting that defective apoptotic programs may contribute to treatment failure, which is commonly seen in most human cancers. Studies have shown that caveolin-1 is over-expressed in colon cancer tissues, as compared to normal colonic mucosa, and colon cancer cells [18–20], suggesting that caveolin-1 may have pro-tumorigenic properties in this type of cancer. However, whether and how caveolin-1 modulates apoptosis of colon cancer cells remains to be determined. An important regulatory step in apoptosis is the activation of the proapoptotic factor Bax, which translocates to the outer mitochondrial membrane and oligomerizes following its activa- tion. Bax promotes apoptosis by making the membrane permeable to cytochrome c. Ku70 is a protein that was originally identified as part of the Ku70/Ku80 complex that mediates the repair of DNA double-strand breaks by non-homologous end joining (NHEJ) [21]. However, Ku70 has also been shown to regulate apoptosis through a Bax-dependent mechanism. Over-expression of Ku70 inhibits Bax-mediated apoptosis, whereas depletion of Ku70 enhances apoptosis [22]. In addition, Ku70 2/2 mice are hypersensitive to ionizing radiation [23] and cells from these mice are also hypersensitive to apoptosis induced by staurosporine [24]. PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39379