activation [22]. Besides, the association between protein phospha- tase 2Cb (PP2Cb) and the IKK complex has been demonstrated [23], and ELKS has also been identified as a new regulatory subunit of IKK complex that mediates the recruitment of IkB to the complex [24]. However, there is no indication of a mitochondrial protein involved in the IKK/NF-kB activation. We have recently identified heat shock protein 60 (Hsp60) as an IKK-interacting protein. Hsp60 is the mitochondrial chaperonin that promotes the folding of the imported protein to native conformation. Nonetheless, the existence and function of Hsp60 in extramitochondrial compartments has frequently been stated [25]. For example, Hsp60 can be a ligand for cell-surface receptors like toll-like receptor [26]. Intracellularly, Hsp60 has been shown to interact with pro-caspase-3 or Bax [27,28,29]. However, the function of Hsp60 related to cell survival signaling has not been reported. In this study, Hsp60 was found to mediate the NF-kB- dependent survival signaling in the cells. Specifically, Hsp60 directly interacted with IKKa/b in cytoplasm and then promoted the phosphorylation-dependent activation of the kinase in response to TNF-a. Such signaling function of Hsp60 was independent of its chaperone activity, which sets Hsp60 apart from other heat shock proteins that function via stabilizing or destabilizing signaling complex [30]. We also showed in vivo evidence that cytosolic expression of Hsp60 protects hepatic cells against chemical-induced damages via enhancing IKK activation. Thus, this finding represents the novel pro-survival function of cytosolic Hsp60 and shed a light on understanding the function of Hsp60 in extra-mitochondrial compartments [25]. Results Hsp60 interacts with IKK complex in cytoplasm To identify an additional component, we examined the molecular composition of the latent IKK complex using a proteomic technique combining immuno-affinity purification and mass spectrometry. Briefly, the IKK complex was precipitated from the lysates of unstimulated HeLa S3 cells using anti-IKKa antibody beads, and the co-precipitated proteins were sequenced by liquid chromatography-tandem mass spectrometry. The identification of the IKK subunits and Hsp90 indicated that the immunopurification of IKK complex fairly worked (Fig. 1A). As a consequence, this proteomic study identified a heat shock protein, Hsp60, in the precipitates (Fig. 1A and 1B). The presence of the IKK subunits and Hsp60 in the precipitates was confirmed by immunoblotting (Fig. 1C). Then, we decided to investigate the biological meaning of the IKK-Hsp60 interaction. The first step was to verify the endogenous interaction of Hsp60 and IKKs by co-immunoprecipitation experiments. When the heterogeneous IKK complexes were precipitated with antibodies against IKKa, IKKb, and IKKc, each of the IKK subunit-specific antibodies similarly precipitated Hsp60 (Fig. 1D). In addition, Hsp90 was also co-precipitated with IKK complex [20,21]. This interaction was found to be unaffected by TNF-a treatment (Fig. 1E), indicating that Hsp60 is a component protein of heterogeneous IKK complexes. A reverse immunoprecipitation was then carried out with the cytosolic fraction to exclude the mitochondrial contamination. The anti-Hsp60 antibodies co- precipitated IKKc with Hsp60, whereas control goat IgG did not (Fig. 1F), confirming that cytosolic interaction of Hsp60 and IKK. In order to visualize the virtual interaction of Hsp60 with IKKs in cytoplasm, the immunogold staining combined with the electron microscopy (EM) was performed. The immune complexes of Hsp60 and IKK with their specific antibodies were detected differently using secondary antibodies labeled with 20 nm- and Hsp60 for IKK Regulation 40 nm-diameter gold particles, respectively. As a result, the Hsp60-labeling gold particles were distributed throughout the cellular structures: not only in the matrix and intermembrane space of mitochondria, but also in the cytoplasm and plasma membrane (Fig. 2B). In contrast, the IKKa- and IKKb-labeling gold particles were mainly detected in the cytoplasm (Fig. 2C and 2D), while the IKKa was also detected in the nucleus, which is consistent with the previous reports [31,32]. Although the IKK core subunits have been shown to be found in the mitochondrial fraction [33], our data showed that the IKK-labeling gold particles were often seen in the vesicular structures rather than the mitochondria (Fig. 2C and 2D). This discrepancy might be due to the previous study done with subcellular fractionation. When the Hsp60 and IKKs were co-stained, the direct binding of 20 nm and 40 nm gold particles was clearly detected in the cytoplasm (Fig. 2E and 2F). It should be noted that not all of the IKKa and IKKb were associated with Hsp60. These results collectively indicate that the Hsp60 directly interacts with IKK complex in the cytosol. Hsp60 directly interact with IKKa/b, not IKKc Next, we analyzed the molecular interaction of Hsp60 and IKKs. To do this, a cytosol-targeted version of Hsp60 (Hsp60c), wherein the mitochondrial targeting signal sequence is deleted, was constructed. When Hsp60c was co-expressed with each of the IKK core subunits, Hsp60c interacted with IKKa and, albeit to a lesser extent, with IKKb, but not with IKKc (Fig. 3A). Then, an in vitro binding experiment using the recombinant proteins of glutathione-S-transferase (GST)-fused Hsp60 and (His)6-tagged IKK core subunits was assessed by a GST pull-down assay. The result, again, indicates that Hsp60 binds directly to IKKa and IKKb, but not to IKKc (Fig. 3B). The molecular interaction of Hsp60 with IKK was further characterized by domain mapping experiments. Because the C- terminal deletion hampered the ectopic expression, a series of N- terminal deletion mutants of Hsp60c was tested for IKK binding via co-expression with Flag-tagged IKKa in HEK293 cells (Fig. 3C). The results showed that the N-terminal part (,160 amino acids from N-terminus) of Hsp60 protein was shown to be dispensable for the interaction (Fig. 3D). The same result was obtained when endogenous IKK complex was immunoprecipitat- ed from HeLa cells transfected with the Hsp60c constructs (Fig. 3E). The results fairly indicate that the core binding domain is located in the middle of Hsp60 protein. Hsp60 is involved in the IKK/NF-kB activation Next, the biological effect of cytosolic Hsp60-IKK interaction was investigated in TNF-a-mediated NF-kB pathway. To achieve the goal, the essential step is to manipulate the level of cytosolic Hsp60 without affecting the mitochondrial one because Hsp60 deficiency is known to cause a mitochondrial functional defect [34,35,36]. Interestingly, a number of studies has previously reported that an antisense oligodeoxynucleotide (AS- ODN) complementary to a sequence surrounding the start codon of the human Hsp60 open reading frame actually reduces the cytosolic Hsp60 level [27,37,38]. We, therefore, decided to test this AS-ODN (designated as AS-1) for a selective knockdown effect. In order to exclude the possibility of non-specific action of a particular ODN sequence, we chose a second AS-ODN (AS-2) that is complementary to the region (+95,+110 from start codon) near the 59 end, but after mitochondrial targeting signal sequence (MTS), of Hsp60 open reading frame (Fig. S1A). The sense ODN (S-ODN) complementary to AS-1 was used as a control ODN. Since the antisense ODN is a moderate PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9422