In this study, we show that GCUNC-45 is a component of PR and hsp90 complexes isolated from cell lysates. Direct in vitro interactions of GCUNC-45 with both isoforms of PR and with hsp90 were shown, and a second, novel TPR-binding site on hsp90 was revealed. We also establish that GCUNC-45 is a positive cofactor for the cellular transcriptional activity of PR, and this appears to involve interplay between GCUNC-45 and the cochaperone FKBP52.
GCUNC-45 has only recently been identified as a protein closely related to SMUNC-45, and it appears to be unique to vertebrates (37
). Because all other members of the UCS family have been implicated in the processing and functions of myosins, GCUNC-45 was proposed to be a myosin chaperone for non-muscle cells that would be involved in cytoskeletal functions (37
). It has been shown to differ from SMUNC-45 in its tissue distribution and pattern of expression during embryogenesis. Antisense experiments indicated that a loss of GCUNC-45 suppressed myotube formation in muscle cells and reduced the rate of proliferation in non-muscle cells (37
). Our results show that GCUNC-45 activities are not confined to myosin interaction. One can even consider the possibility that the myosin-binding activity might be important to the PR chaperone complex, perhaps as a means for localization or trafficking of the complex.
The presence of potential NR box motifs initially suggested that GCUNC-45 might be a transcriptional coregulator of PR. We have not excluded this possibility, but its primary localization in the cytoplasm and its association with hsp90 indicates a role in PR chaperoning. We have found that the LXXLL sequences are not essential for binding of GCUNC-45 to PR in vitro, although their mutation reduces binding somewhat (results not shown).
UNC-45 from Caenorhabditis elegans
has been reported to interact with hsp90 through its TPR motifs (1
). This interaction appeared to involve the C terminus of hsp90, based on competition by a peptide containing the C-terminal sequence MEEVD. Because of these results and the proposed mechanism for the interaction of hsp90 with UNC-45 (1
) and other cochaperones (6
), it seemed likely that the TPR recognition motif in the C terminus of hsp90 would be involved in the interaction with GCUNC-45. However, instead, a new site within the N-terminal ATP-binding domain of hsp90 was revealed. The existence of a second TPR-binding site on hsp90 opens new possibilities for identifying factors that might bind to this site to regulate hsp90 functions and may stimulate a reassessment of known chaperone-cochaperone interactions. Perhaps some cochaperones can recognize either TPR-binding site depending on the conformational state of hsp90. An intriguing parallel may exist with the complex of Hip-hsp70-Hop. Like hsp90, hsp70 has an EEVD sequence at its C terminus which appears to be part of the Hop-binding domain near the C terminus of hsp70 (5
). However, while the cochaperone Hip binds hsp70 through its TPR domain, it binds to a site, yet to be identified, in the N-terminal ATPase domain of hsp70 (10
GCUNC-45 has a positive role in the hsp90 chaperoning pathway for PR. In the cell, its overexpression caused only a small enhancement of PR transcriptional activity to induce a reporter gene, but the loss of endogenous GCUNC-45 resulted in a marked reduction of PR activity. This reduction of PR activity appears linked to the loss of GCUNC-45, since the protein levels of PR, hsp90, hsp70, Hop, and FKBP52 were not altered by the treatment. However, in simplified cell-free systems, GCUNC-45 inhibits the ATPase activity of hsp90 and blocks progression of PR chaperoning to its hormone-binding state. We have only observed the inhibition of ATPase activity when this activity was enhanced by the presence of Aha1. Aha1 has been shown to bind to the middle domain of hsp90 and is thought to facilitate repositioning of a catalytic loop of this domain that interacts with the γ phosphate of ATP bound in the N-terminal domain (28
). Thus, the binding of GCUNC-45 to hsp90 may interfere with the conformational changes needed to bring the catalytic loop of the middle domain into contact with bound ATP, or it may block the binding of Aha1 to hsp90 even though the two proteins do not seem to share a common binding site.
Since ATP binding and hydrolysis are essential to the activity of hsp90 (14
), their inhibition explains the loss of proper PR chaperoning in the presence of GCUNC-45 as illustrated by a loss of hormone-binding activity. However, this appears not to be the case in the complete chaperoning system of the cell. The cochaperone Hop is also a potent inhibitor of the hsp90 ATPase activity, but this inhibition is confined to one step in the chaperoning process and is reversed by the association of Hop-hsp90 with hsp70 (17
). As a result, Hop has a very positive effect on the chaperoning activity of hsp90. The cochaperone Cdc37 is essential for the chaperoning of many protein kinases, yet it inhibits the ATPase activity of hsp90 in vitro (45
). It has been suggested that this inhibited state is more optimal for initial interaction of hsp90 with client proteins. In like manner, we propose that GCUNC-45 enters the chaperone complex at an intermediate stage where its inhibitory action serves a purpose, but that this inhibition is relieved by subsequent events that are missing from the in vitro system (Fig. ). One such event is the binding of the immunophilin FKBP52 in the PR complex. This protein is observed in PR complexes from cell lysates and appears to interact with both PR and hsp90. Its function is still unclear, but it may be involved in modulating PR conformation or trafficking (35
). Future studies may reveal additional interactions between GCUNC-45 and other hsp90 cochaperones and clients.
FIG. 8. Model for PR chaperoning showing a transient association of GCUNC-45 during the progression from the intermediate to the mature complex. FKBP52, and possibly other cochaperones that bind hsp90 through TPR domains, may displace GCUNC-45 from PR complexes (more ...)