Facilitating Akt Clearance via Manipulation of Hsp70 Activity and Levels

Abstract

Members of the 70-kDa heat shock family can control and manipulate a host of oncogenic client proteins. This role of Hsp70 in both the folding and degradation of these client proteins makes it a potential drug target for certain forms of cancer. The phenothiazine family of compounds, as well as the flavonoid myricetin, was recently shown to inhibit Hsp70-ATPase activity, whereas members of the dihydropyrimidine family stimulated ATPase function. Akt, a major survival kinase, was found to be under the regulation of Hsp70, and when the ATPase activity of Hsp70 was increased or decreased by these compounds, Akt levels were also increased or decreased. Also, increasing Hsp70 levels concurrent with inhibition of its ATPase function synergistically reduced Akt levels to a greater extent than either manipulation alone, providing new insights about client fate decisions. Akt reductions mediated by Hsp70 inhibitors were prevented when Hsp70 expression was silenced with small interfering RNA. Inhibiting Hsp70 ATPase function produced cytotoxic events only in breast cancer cell lines where Akt dysfunction was previously shown, suggesting therapeutic specificity depending on the Hsp70 client profile. Thus, increasing Hsp70 levels combined with inhibiting its ATPase function may serve to dramatically reduce Akt levels and facilitate cell death in certain types of cancer.Heat shock proteins are the primary regulators of protein folding and degradation in the cell. The ATPase activity of Hsp70 and Hsp90 variants serves as the hub whereby all other chaperones and co-chaperones are activated, either to facilitate recycling or for clearance of chaperone substrates or clients. Much of the effort surrounding chaperone research has focused on either altering the expression levels of these heat shock proteins by activating the heat shock transcription factor (HSF1) or by inhibiting the activity of Hsp90 (1, 2). Small molecules have been identified that can produce either or both of these outcomes (3, 4). However, despite its clear role in a number of diseases of aging, very little emphasis has been placed on the enzymatic ATPase activity of Hsp70. To address this deficiency, we recently identified several compounds that could either stimulate or inhibit Hsp70 ATPase function without affecting its levels (5). These compounds bind to Hsp70 allosterically, affecting the rate of ATP consumption, which is tied to opening and closing of the protein lid over the substrate binding domain. When ATP is converted to ADP, the lid closes, preventing release of the client. When ADP is exchanged for ATP, the lid opens, and the client is released. We demonstrated that Hsp70 inhibitors force this lid to stay open, whereas activators increase the rate of lid opening and closing (5). With these compounds defined, we are now able to compare the effects of increasing Hsp70 expression levels with modulating its ATPase function for distinct disease-related clients.With this in mind, and based on previous findings that the prosurvival kinase, Akt, was a wel...

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