Hsp60 for IKK Regulation Figure 3. Hsp60 directly interacts with IKK complex. A. Direct binding of Hsp60 and IKK subunits. The 293T cells were co-transfected with Hsp60c (HA tag) and each of IKK subunit proteins (Flag tag) for 24 hrs. B. In vitro association of Hsp60 with IKKa and IKKb. GST-fused Hsp60 proteins bound to the glutathione Sepharose beads was incubated with the lysates of Sf9 insect cells expressing His6-tagged IKK proteins. Hsp60 and IKKs were detected by immunoblotting for GST and HA tags, respectively. C. Schematic diagram showing deletion mutants of Hsp60. The putative phosphorylation sites of kinases, including PKA/PKG (1) and PKC (2), are indicated. D and E. Interaction of Hsp60 wild-type (WT) and deletion mutants with ectopically-expressed IKKa in 293T cells (D) or to endogenous IKK complex in HeLa cells (E). The control vector (C) is indicated. Representative blots and images are shown (n = 3). n.s., nonspecific. doi:10.1371/journal.pone.0009422.g003 using the loss-of-function mutants strongly suggest that the cytosolic Hsp60 functions independently of chaperone activity in IKK/NF-kB activation. One of the IKK-interacting protein, ELKS, has been shown to mediate the IkB recruitment to IKK complex [24]. To test this mode of action, the recombinant Hsp60 protein was directly added into the IKK kinase reaction, where the activated IKK complex is incubated with full-length human IkB as a substrate. The in vitro kinase activity of the activated IKK toward IkB was not affected by the presence of Hsp60 protein (Fig. 7D), indicating that Hsp60 is not involved in the interaction of IKK and its substrate IkB. Lastly, a direct involvement of cytosolic Hsp60 in IKK activation was addressed by examining the activation-dependent serine phosphorylation in the T-loop of IKKa/b. The AS-ODN transfection markedly abolished the TNF-a-induced phosphory- lation of IKK at Ser178/181, indicating that the phosphorylation- dependent IKK activation was impaired (Fig. 7E). Conversely, the ectopic expression of Hsp60c resulted in an increase of IKK phosphorylation (Fig. 7F). Overall, the data indicate that the cytosolic Hsp60 is involved in the phosphorylation-dependent IKK activation, rather than the chaperone-dependent stabilization of IKK complex. Cytosolic Hsp60 affects the NF-kB target gene expression and cell survival To determine the significance of cytosolic Hsp60-mediated regulation of the IKK/NF-kB pathway, we examined the expression of NF-kB target genes in ODN-transfected cells. When the expression of anti-apoptotic genes was screened by a RNase protection assay [44], the expression of TRAF1, c-IAP1, and c- IAP2 were not affected by AS-ODN transfection (Fig. 8A). This was unexpected but led us to postulate a possibility that some select target genes related to mitochondrial protection can be affected. To test this, we looked at the induction of several target genes, including antioxidant proteins (ferritin heavy chain and MnSOD) and Bcl-2 members (Bcl-2, Bcl-XL, Bfl-1/A1). Interestingly, the AS-ODN significantly diminished the induction of only MnSOD and Bfl-1/A1 expression in response to TNF-a (Fig. 8B). The Hsp60N antibody also significantly reduced the induction of these genes (Fig. 8C). It was again confirmed that the induction of the c- IAP2 expression was not affected in either case. Thus, the results PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9422