Caveolin-1, a Novel Ku70-Interacting Protein Figure 3. Ku70 binds to the scaffolding domain of caveolin-1. (A) The consensus caveolin binding domain (CBD) is shown together with the caveolin binding domain of Ku70 (amino acids 471 to 478) and a mutant form of Ku70’s CBD in which aromatic residues were mutated to alanines. (B) Ponceau S staining of GST alone and GST-caveolin-1 fusion proteins. (C) GST-caveolin-1 fusion protein pull-down assays were performed using cell lysates from HCT116 cells transiently transfected with wild type Ku70-HA. A blot that is representative of two independent experiments is shown. doi:10.1371/journal.pone.0039379.g003 The Interaction between Caveolin-1 and Ku70 is Induced by Chemotherapeutic Drugs and Limits the Release of Bax from Ku70 Once we demonstrated that Ku70 was a novel caveolin-1- interacting protein, we then asked whether binding of caveolin-1 to Ku70 was the molecular mechanism underlying the anti-apoptotic properties of caveolin-1. HCT116 cells were treated with either 5- FU or etoposide and the interaction between caveolin-1 and Ku70, before and after treatment with chemotherapeutic drugs, was evaluated by coimmunoprecipitation studies. We show in Figure 4A that the interaction between caveolin-1 and Ku70 was marginal under resting conditions but dramatically increased upon treatment with either 5-FU or etoposide. Consistent with these data, Ku70 was mainly localized in the nucleus and, to a lesser extent, diffuse in the cytoplasm (see arrowheads in Figure 4B) in HCT116 cells under resting conditions, away from caveolin-1, which was enriched at the plasma membrane (Figure 4B). In contrast, although nuclear localization was still evident, Ku70 was found to partially co- localize with caveolin-1 at the plasma membrane and intracellular caveolar membranes after 5-FU treatment (see arrows in Figure 4B). Interestingly, 5-FU, but not etoposide, also stimulated total caveolin- 1 protein expression in HCT116 cells (Figure 4A, lower panel). This is consistent with the increased caveolin-1 mRNA level induced by 5-FU, but not etoposide, in HCT116 cells (Figure S5A), suggesting that 5-FU also promotes caveolin-1 protein expression through a transcriptional-dependent mechanism. Why does the chemotherapeutic drug-induced interaction between caveolin-1 and Ku70 act in an anti-apoptotic manner? Ku70 has been shown to bind to Bax under resting conditions. Release of Bax from Ku70 and the translocation of Bax to mitochondria are key steps in the signaling events that link an apoptotic stimulus to cell death. The molecular mechanisms that regulate these processes remain to be fully elucidated. Our data show that caveolin-1 acts as an anti-apoptotic protein (Figures 1 and 2) and chemotherapeutic drugs, which induce apoptosis, promote the interaction between caveolin-1 and Ku70 (Figures 4A and 4B). To investigate the functional significance of the binding of caveolin-1 to Ku70, we tested the hypothesis that binding of caveolin-1 to Ku70 inhibits apoptosis by limiting the release of Bax from Ku70. HCT116 cells were transfected with either scrambled or caveolin-1 siRNA and treated with either 5-FU or etoposide. Protein cell lysates were then immunoprecipitated with an antibody probe specific for Bax and immunoprecipitates subjected to immunoblotting analysis with anti-Ku70 IgGs. Figure 5A shows that both 5-FU and etoposide stimulated the release of Bax from Ku70 in scrambled siRNA- transfected cells. Importantly, the dissociation of Bax from Ku70 was potentiated in cells lacking caveolin-1. Thus, the interaction of caveolin-1 with Ku70 limits the release of Bax from Ku70 induced by chemotherapeutic drugs. In support of the existence of a caveolin- 1/Ku70/Bax complex after treatment with chemotherapeutic drugs, we show in Figure 4A that both 5-FU and etoposide promoted the interaction between caveolin-1 and Bax, and in Figure 5B that caveolin-1, Ku70 and Bax are all found by immunoblotting analysis after immunoprecipitation of protein cell lysates from 5-FU-treated HCT116 cells with antibody probes specific for either caveolin-1 or Bax. Knockdown of Caveolin-1 Protein Expression Potentiates the Chemotherapeutic Drug-induced Conformational Change of Bax Activation of Bax is associated with a conformational change that leads to exposure of the N terminus and can be detected using the anti-Bax antibody clone 6A7 [29,30]. We show in Figures 5C and 5D that 6A7 antibody immunoprecipitated Bax only in HCT116 cells treated with the apoptotic stimulus 5-FU but not in control cells. Knockdown of caveolin-1 protein expression by siRNA significantly potentiated binding of the 6A7 antibody to Bax in cells treated with 5-FU (Figures 5C and 5D). However, PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39379