Chaperone Activity and Dimerization Properties of Hsp90 and Hsp90 in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery.

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90 and Hsp90, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90 and Hsp90 in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90 and Hsp90 have equivalent chaperone activities, and when mixed together in this assay, they formed only GR•Hsp90 and GR•Hsp90 homodimers and no GR•Hsp90 heterodimers. In contrast, GR•Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GR•Hsp90 heterodimers. The formation of GR•Hsp90 heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90 heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90 at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90 T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90 in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90 chaperone activity.