TPR domain-containing proteins participate in a variety of cellular functions, and whereas many of these proteins do not interact with heat shock proteins, a specific subset does. As demonstrated here, CHIP is a previously undescribed cytoplasmic protein that can complex with Hsc70 and Hsp70. CHIP is distinguished by the degree to which its amino acid sequence is conserved across species, from Drosophila to humans. As expected, the three TPR domains are highly conserved (70% similarity across all three species); even more remarkable is the similarity across species in the carboxy-terminal region (87% similarity from amino acids 210 to 303 of the human sequence). The CHIP carboxy-terminal domain is not similar in sequence to that of any other protein known to interact with heat shock proteins, such as cyclophilins or molecular chaperones, making it likely that the cellular function of CHIP is unique among heat shock protein-interacting proteins.
Examination of the tissue distribution of CHIP mRNA reveals notable differences in its expression. In the adult human, expression is highest in striated muscle (skeletal muscle and heart), less in the pancreas and brain, and negligible in other tissues. Common features of the highly expressing organs include a large proportion of terminally differentiated, nonproliferating cells and relatively high levels of metabolic activity. CHIP is also expressed in a developmentally and spatially regulated fashion in the mouse embryo, particularly in the developing nervous system and during the course of cardiac and skeletal myogenesis (28a
). The functional significance of the tissue-restricted expression pattern of CHIP in vivo is not clear at present, although it is possible that CHIP is associated in some way with developmental processes in the nervous system and muscle. An appreciation for the general role of Hsc70-related proteins in the control of developmental processes (for example, regulation of WT1 activity) is emerging (1
The presence of multiple TPR domains in CHIP suggested to us that CHIP participates in interactions with other proteins. We used the yeast two-hybrid system to define potential CHIP binding partners in vivo, and we found both Hsc70 and Hsp70 to be the most frequently identified interaction partners. There is concern that interactions with Hsp70 or Hsc70 in this assay may reflect a nonspecific interaction with bait proteins that misfold in yeast; interactions of this type would represent Hsc70-Hsp70 chaperone activities rather than functional protein-protein interactions. However, four lines of evidence suggest that this is not the case: (i) these interactions are also detected in vitro with bacterially expressed and in vitro-translated CHIP; (ii) the interaction domain of CHIP contains TPR motifs similar to those of other members of heat shock protein-associated complexes; (iii) CHIP interacts with the carboxy-terminal domain of Hsp70 and Hsc70 in vitro and in vivo, rather than with their substrate-binding domains, which are known to interact with misfolded proteins (5
); and (iv) CHIP-Hsc70 complexes can be detected in vivo by immunoprecipitation. Based on the interactions observed in vitro and in vivo, we consider CHIP to be a bona fide interaction partner with Hsp70 and Hsc70.
A number of other proteins interact with Hsp70 and Hsc70 to regulate their cellular functions. Hip and BAG-1 interact with the ATPase domain to stabilize ADP binding and stimulate its release, respectively (17
), and auxilin binds the ATPase domain to assist in uncoating clathrin-coated vesicles (36
). In addition to mediating homo-oligomeric interactions of Hsc70 and Hsp70 (2
), the 10-kDa carboxy-terminal domain (amino acids 537 to 646 of Hsc70) provides noncompetitive and probably independent binding sites for Hop and Hsp40 (8
). Hop serves as an organizing factor mediating the interactions between Hsp70 and Hsp90 (6
), whereas Hsp40 is a cochaperone that enhances ATPase activity and stable associations with unfolded peptides (11
). We now demonstrate that the carboxy-terminal domain is both necessary and sufficient for interactions between CHIP and Hsp70-Hsc70, making CHIP the third member of the group of known carboxyl terminus-binding proteins. We do not yet know whether CHIP interacts with either the Hsp40 or Hop binding site or whether there is a distinct binding site for CHIP in the carboxy-terminal domain. Both Hop and CHIP are TPR domain-containing proteins, which suggests that they may interact with Hsc70 and Hsp70 through common mechanisms; however, it is equally plausible that CHIP interacts in some way with the Hsp40 binding site, since the biochemical properties of CHIP could be explained by interference of Hsp40 binding with Hsc70.
It is notable that CHIP, which binds to the carboxyl termini of Hsc70 and Hsp70, shows strong structural similarities with TPR domain-containing proteins (Hip, Cyp-40, and protein phosphatase 5) that interact with Hsp90 or with the ATPase domains of Hsc70 and Hsp70 (Fig. C). This finding suggests that TPR motifs of proteins that interact with members of the heat shock protein family form a structurally related subset within the larger family of TPR-containing proteins. If this is indeed the case, what then determines the specificities of interactions with heat shock proteins? Although the answer to this question is likely to be determined by structural comparisons, it is worth noting that the TPR domains of CHIP alone are necessary but not sufficient for interactions with Hsc70 and Hsp70 (Fig. ) and that an adjacent charged domain is also required. Since a positively charged domain adjacent to the TPR domain of Hip is similarly required for interactions with the ATPase domain of Hsc70 (18
), it is possible that the binding specificities for this class of TPR-containing proteins are determined by sequences adjacent to the TPR domains.
Previously, three proteins have been shown to regulate the Hsc70-Hsp70 substrate-binding cycle. Hsp40 enhances the forward reaction of this cycle by increasing Hsc70 ATPase activity and thus promoting the formation of the ADP-bound, high-substrate-affinity Hsc70 conformation (26
). Hip stabilizes ADP-bound Hsc70 (17
); therefore, both Hip and Hsp40 enhance Hsc70-mediated refolding. The antiapoptotic protein BAG-1 has been shown recently to facilitate the reverse reaction by promoting the exchange of ATP for ADP and release of substrate (16
); BAG-1 therefore opposes the actions of Hip and attenuates Hsc70 chaperone activity. Whereas Hip might be considered to antagonize the reverse reaction of the Hsc70 cycle, a negative regulator of the forward reaction has not been described. Based on its ability to decrease the ATPase activity of Hsp70 and Hsp40, CHIP is the first described candidate protein to inhibit the forward reaction cycle. Consistent with this role, CHIP inhibits luciferase refolding and substrate binding that occurs in the presence of Hsp70 and Hsp40, although CHIP has no effect by itself in either of these assays. Based on these results, a model can be proposed whereby CHIP and BAG-1 act on the forward and reverse Hsp70-Hsc70 reaction cycles, respectively, to maintain the low substrate affinity conformation; in contrast, Hip and Hsp40 coordinately regulate the two cycles to favor the ADP-bound state (Fig. ). Potential molecular mechanisms for the effects of CHIP on the reaction cycle include prevention of ATP binding or inhibition of ATP hydrolysis; future studies will be needed to address these possibilities.
FIG. 10 Model of the eukaryotic reaction cycle in the presence of CHIP, Hsp40, Hip, and BAG-1. The forward reaction, in which ATP is hydrolyzed to ADP and inorganic phosphate (Pi) is released, is enhanced by Hsp40. The biochemical data suggest that CHIP blocks (more ...)
At present, it is unclear whether the effects of CHIP on the Hsp70-Hsc70 substrate-binding cycle are mediated solely through the TPR and charged domains (perhaps by competing with or otherwise altering the affinity of Hsp40 for binding to Hsc70) or whether the highly conserved carboxy-terminal domain also regulates Hsc70-related functions in some way. The three proteins sharing similarity with this domain—UFD2, NOSA, and the p40 subunit of the 26S proteasome—have functions linked to the ubiquitin-proteasome pathway (9
). Although a function has not yet been ascribed to the similar region in any of these proteins, it is tempting to speculate that CHIP may serve in some way to mediate interactions between the chaperoning and ubiquitin-proteasome systems, as Hsc70 and Hsp70 are required for ubiquitin-dependent degradation of some, but not all, protein substrates (3
). The amino acid sequence similarities between proteasome-associated proteins and CHIP, a regulator of the Hsc70-Hsp70 substrate-binding cycle, emphasize the need to define better the relationship between chaperoning and proteolysis.