Hsp60 for IKK Regulation Figure 7. Cytosolic Hsp60 regulates IKK phosphorylation independently of chaperone activity. A. Association of Hsp60c wild-type (WT) and mutants with IKKa and IKKb. The indicated proteins were co-expressed in 293T cells as shown in Fig. 3A. B and C. IKK (B) and NF-kB transcriptional activation (C) in cells expressing Hsp60c wild-type and chaperone-inactive mutants. The kinase and reporter activities were analyzed as described in Fig. 4 (for reporter assay, n = 6, *P,0.0001 versus unstimulated counterpart). D. In vitro kinase activity of IKK in the presence of recombinant Hsp60 protein. The IKK complex was immunoprecipitated from HeLa cell lysates and incubated with or without the indicated GST proteins (20 mg each) in the kinase reaction buffer for 10 min before the kinase reaction. E. Serine phosphorylation of IKKa/b in HeLa cells transfected with AS-1 ODN. Data in the graph are means 6 S.D. (n = 3, *P,0.02, *P,0.001). F. Serine phosphorylation of IKKa/b in Hsp60c-expression HeLa cells. A representative blot is shown (n = 3). doi:10.1371/journal.pone.0009422.g007 study targets MTS-encoding region of Hsp60 mRNA transcript but reduces the level of cytosolic Hsp60 is also supportive. Hsp60 once located in the cytosol exhibited the faster turnover compared to the mitochondrial one. Its rapid turnover is controlled in a proteasome-dependent manner, but it is unlikely due to the structural instability because the cHsp60 protein lacking MTS sequence was successfully expressed and purified in soluble form. Nonetheless, since we cannot exclude the possibility of natural mitochondrial targeting error, the investigation on how mitochondrial Hsp60 is released and whether the turnover of cytosolic Hsp60 depends on ubiquitination is on-going. Cytosolic Hsps such as Hsp70 and Hsp90 are known to broadly participate in cell signaling [30]. As a chaperone protein, they stabilize or destabilize the large signaling protein complexes. Since the interaction of Hsp60 and IKK complex was unaffected by TNF- a stimulation, the mode of Hsp60 action was initially thought to be the same. However, our results shown suggest a unique feature of the signaling function of cytosolic Hsp60. Briefly, the polymeriza- tion-defective or ATPase-inactive mutant of Hsp60 enhanced IKK/ NF-kB activation much more effectively than the wild-type Hsp60 did (see Fig. 8). This suggests that Hsp60 likely functions as a non- chaperonic monomer rather than a chaperone polymer in augmenting NF-kB activation. Some experimental evidences also favorably support the possibility of non-chaperonic function of cytosolic Hsp60: 1) the introduction of the Hsp60N neutralizing antibody did not affect the amount of IKK core subunits in the lysate and immunoprecipitate, and 2) the AS-ODN did not affect the amount of IKK core subunits in the high-molecular-weight fractions (.600 kDa) from gel filtration chromatography (data not shown). These results indicate that the cytosolic Hsp60 is not involved in the stability of the IKK complex. Rather, our study clearly demonstrates that cytosolic Hsp60 is involved in the regulation of IKK phosphorylation in the activation T-loop. Therefore, the cytosolic Hsp60 may act on either enhancing the interaction of the IKK complex with an upstream kinase or the oligomerization-dependent auto-phosphorylation [52]. In any case, it can be a novel mechanism of the IKK activation in collaboration with the ubiquitin-dependent mechanism. In summary, cytosolic presence of Hsp60 is frequently detected and play signaling roles in the cell types, e.g. cardiac myocytes and hepatocytes [37,62,63]. And also there are attempts to approach anti-cancer therapy through understanding the presence and PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9422