Hsp60 for IKK Regulation Figure 2. Visualization of Hsp60 and IKK interaction at a single-cell level. HeLa cells were immunoreacted with no primary (A), anti-Hsp60 (B), anti-IKKa (C), anti-IKKb (D), anti-Hsp60/IKKa (E), and anti-Hsp60/IKKb (F) antibodies, and then labeled with the corresponding secondary antibodies conjugated with 20 nm or 40 nm gold particles, as described in Experimental Procedure. The labeling was assessed by immuno-gold electron microscopy. Nuclei (Nu) and mitochondria (M) are indicated. Arrows indicate direct adherence of Hsp60- and IKK-labeled gold particles. No immunoreactive signal was seen in the sample without primary antibodies (A). The experiments were repeated twice with the same results, and representative results are shown. doi:10.1371/journal.pone.0009422.g002 A similar study was performed by blocking the cytosolic Hsp60 using a specific antibody (Hsp60N), which has been used for immunoprecipitation and immunostaing of Hsp60 (see Fig. 1). The antibody transduction was achieved by a peptide-mediated protein delivery system [39]. The control goat IgG and Hsp60N antibody were found to be successfully delivered to cytoplasm, as being not merged with Mitotracker (Fig. 5A), and Hsp60N, but not control IgG, bound to Hsp60 (Fig. 5B). This result indicates that the delivered antibody can act as a function blocker. Then, IKK/NF-kB activation was examined in antibody-transduced cells. The Hsp60N antibody evidently reduced the IKK activation in response to TNF-a by 50% of the level obtained with the control IgG (Fig. 5C). In contrast, TNF-a-induced JNK activation was not affected, which again proves that the role of Hsp60 is specific to the IKK activation. Consistently, the Hsp60N antibody significantly reduced the transcriptional activity of NF-kB (Fig. 5D). The data collectively conclude that cytosolic Hsp60 promotes the TNF-a-induced IKK/NF-kB signaling. Ectopic expression of cytosol-targeted Hsp60 sufficiently promotes IKK/NF-kB activation Conversely, the role of cytosolic Hsp60 in IKK/NF-kB pathway was addressed by over-expression of cytosol-targeted Hsp60c. The ectopically-expressed Hsp60c was found to associate with the IKK complex (Fig. 6A) and markedly enhanced the IKK and NF-kB activation in response to TNF-a (Fig. 6B and 6C). It should be noted that the ectopic expression of Hsp60c marginally induced the basal IKK and NF-kB activation. The effect of Hsp60c expression in NF-kB activation was completely abolished in IKKb-deficient cells (Fig. 6D), indicating that the regulatory activity of cytosolic Hsp60 is IKK-dependent. In addition, the ectopic expression of Hsp60c did not enhance either JNK activation or the activation of other transcription factors such as AP-1, CRE, and NF-AT (Fig. S2). This result indicates that increasing the cytosolic Hsp60 level augments TNF-a-induced IKK/NF-kB activation. Hsp60 regulates IKK phosphorylation at the activation T- loop To understand the mechanism underlying the regulatory action of Hsp60 in IKK/NF-kB activation, several experimental approaches were attempted. To determine whether the chaperone activity of Hsp60 is required, the two amino acid residues that are known to be critical for the chaperone activity of Hsp60 were considered. One is a lysine residue (K28), which is involved in the oligomerization of Hsp60 protein [40,41]. The other is an aspartate residue (D423), which is an active site residue for ATPase activity [42,43]. Thus, the Hsp60c mutants, wherein K28 and D423 are substituted with glutamate and alanine respectively, were constructed. The co-transfection experiment showed that both mutants interacted with IKKa and IKKb as well as or perhaps even better than the wild type (Fig. 7A). The IKK activation in response to TNF-a in the Hsp60 mutant-expressing cells was similar to that in the wild type (Fig. 7B), indicating that such loss-of-function mutations did not affect the IKK-enhancing activity. Furthermore, TNF-a-induced NF-kB transcription was even enhanced in the Hsp60 mutant-expressing cells at approx- imately 4–6 times higher than that in the vector control (Fig. 7C). The enhancing effect of the mutants was clearly IKKb-dependent, as tested again in IKKb-deficient 3T3 cells. Thus, this experiment PLoS ONE | www.plosone.org 4 March 2010 | Volume 5 | Issue 3 | e9422