ATPase activity of the heat shock protein Hsp72 is dispensable for its effects on dephosphorylation of stress kinase JNK and on heat‐induced apoptosis

Abstract

A major inducible heat shock protein, Hsp72, has previously been found to stimulate dephosphorylation (inactivation) of stress kinase JNK in heat-shocked cells and protect them from apoptosis. Using Rat-1 fibroblasts with constitutive expression of a human Hsp72 or its deletion mutant lacking an ATPase domain (C-terminal fragment (CTF)), we tested whether ATPase activity of Hsp72 is necessary for these effects. We found that expression of CTF markedly increased, similarly to the intact protein, JNK dephosphorylation in heat-shocked cells. As a result, JNK inactivation following heat shock occurred much faster in cells expressing either full-length or mutant Hsp72 than in parental cells and this was accompanied by suppression of heat-induced apoptosis. Thus, protein refolding activity of Hsp72 appears to be dispensable for its effect on JNK inactivation and apoptosis.z 1999 Federation of European Biochemical Societies.

Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

“…In agreement with studies by Li et al and Volloch et al, our results clearly show that Hsp72 protects cells from heatinduced apoptosis and heat-induced protein damage by distinctly different mechanisms (Li et al 1995;Volloch et al 1999). While the repair of heat-denatured luciferase required the chaperone activity of wild-type Hsp72, the substrate-binding domain of Hsp72 was sufficient to inhibit heat-induced apoptosis.…”

“…Our results confirmed that the Hsp72 substrate-binding domain is sufficient to inhibit heat-induced apoptosis in L929.3 cells as described previously in Rat-1 fibroblasts (Volloch et al 1999). Both the 381-640 Δ-ATPase mutant and the K71E point mutant protected cells in an equivalent fashion to the full length protein, while cells expressing the 1-611 fragment, lacking an active substrate-binding domain, apoptosed at an equivalent rate to base vector control cells.…”

“…This study neatly illustrates that the inhibition of cell death does not always require the chaperone repair function of Hsp72. While Li et al measured cell death rather than apoptosis, other studies have reported that the substrate-binding domain is sufficient to inhibit apoptosis (Sun et al 2006;Volloch et al 1999). In the absence of any confirmed antiapoptotic Hsp72-binding targets, the mechanism of how Hsp72 inhibits apoptosis may provide vital clues to how this protein protects cells from stress.…”

“…In each of these examples, Hsp72 was expressed under control of the inducible tetracycline promoter, while those studies that reported protection independent of chaperone activity used constitutive Hsp72 expression systems Sun et al 2006;Volloch et al 1999). Differences in behavior between constitutively and inducibly expressed Hsp72 have been noted previously but never adequately explained.…”

“…Is Hsp72 chaperone activity required for the remodeling of an apoptotic signaling complex in the same way it is required for remodeling the progesterone receptor (Kosano et al 1998)? Mosser et al (2000 reported that chaperone activity was required for the inhibition of apoptosis, while other authors have concluded that only the substrate-binding domain is required for protection Sun et al 2006;Volloch et al 1999). In this study, we expressed several different mutant forms of Hsp72 in cells and compared the ability of these proteins to inhibit apoptosis and promote the refolding of heat labile luciferase.…”