The Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts.

In the two decades that have elapsed since the molecular chaperone Hsp902 was shown to regulate the function of steroid receptors (1), >200 signaling proteins have been found to be regulated by Hsp90 (2). These Hsp90 “client” proteins form complexes containing Hsp90 and Hsp70 that are assembled by a multichaperone machinery (3), with Hsp90 regulating both signaling protein function and turnover. Degradation of these Hsp90-regulated signaling proteins occurs via the ubiquitin-proteasome pathway (4), which in this case is initiated by Hsp70-dependent E3 ligases, such as CHIP and parkin (5). Formation of a complex with Hsp90 stabilizes the client signaling protein, and treatment with a specific inhibitor of Hsp90, such as geldanamycin, triggers its rapid degradation (6). Because many of the Hsp90-regulated signaling proteins are involved in cancer cell growth, Hsp90 inhibitors have emerged as a promising new class of anticancer drugs (7). In this Minireview, we provide a mechanistic basis for understanding how the abundant and ubiquitous chaperones Hsp90 and Hsp70 function together as essential components of the Hsp90 chaperone machinery to regulate signaling protein function and turnover. Like other chaperones, Hsp90 alone has been shown in vitro to assist the refolding of partially unfolded proteins to a properly folded, active conformation. However, Hsp90 is not required for de novo protein folding (8), and it is likely that in cells Hsp90 acts only in concert with Hsp70 in the multichaperone machinery. In contrast to the in vitro experiments on unfolded substrates, this Hsp90 machinery acts on proteins that are in their native conformations to assist the opening of ligand binding clefts. These clefts are hydrophobic clefts that must open to allow access of ligands, such as steroids, ATP, and heme, to their binding sites within the protein's interior. In the absence of the chaperone machinery, ligand binding clefts are dynamic, shifting to varying extents between closed and open states. When clefts open, hydrophobic residues of the protein's interior are exposed to solvent, and continued opening may progress to protein unfolding. Therefore, the extent to which the ligand binding cleft is open determines ligand access and thus protein function, but clefts are inherent sites of conformational instability. The chaperone machinery assists cleft opening, and Hsp90 binding stabilizes the open state of the cleft, preventing further unfolding and Hsp70-dependent ubiquitination. The Hsp90 client proteins are assembled into complexes with the chaperone that are stable enough to be isolated and analyzed biochemically. Although we will refer to these as “stable” Hsp90 complexes, they are constantly undergoing cycles of assembly and disassembly in the cytoplasm and nucleoplasm (3). We will refer to this client protein cycling with Hsp90 as stable cycling. As we will show, a variety of manipulations, including mutations of the LBD or ligand binding itself, result in heterocomplexes that very rapidly disassemble such that no (or only trace amounts of) Hsp90 heterocomplexes can be observed in cell lysates. This rapid complex disassembly we define as “dynamic” Hsp90 cycling, and some signaling proteins naturally interact with Hsp90 in this dynamic cycling mode. Because the function and turnover of these proteins are not as affected by Hsp90 inhibitors as proteins undergoing stable Hsp90 complex assembly, they have not been considered as Hsp90-regulated client proteins, but they are nevertheless Hsp90 substrates. There are several examples where the LBDs of signaling proteins with this dynamic “kiss-and-run” interaction with Hsp90 have been converted by mutation to metastable clefts that undergo stable Hsp90 complex assembly. This conversion of signaling protein-Hsp90 interaction is associated with the acquisition of stringently Hsp90-regulated behavior, typical of client proteins. As Neckers and colleagues have noted (9), many “nodes” in overlapping signaling pathways involved in cancer cell growth are subject to stringent Hsp90 regulation. These Hsp90 client proteins may have evolved from a wide variety of signaling proteins that undergo a more common dynamic cycling of Hsp90 with ligand binding clefts. However, there is no motif for Hsp90 binding, and the basis for its interaction with proteins to form stable or dynamic complexes has not been defined. Here we will present selected examples of Hsp90 effects on signaling protein function and turnover to develop a model in which ligand binding clefts are the common feature determining the interaction with the chaperone. Additional examples in support of the model are cited elsewhere (10).