Binding of immunophilins, protein phosphatase 5, and p50(cdc37) to hsp90 and hsp90-chaperoned signaling proteins.

Abstract

Unliganded steroid receptors exist in heterocomplexes with hsp90 and a tetratricopeptide repeat (TPR) protein, such as an immunophilin or protein phosphatase 5 (PP5). In this thesis, I determined the stoichiometry of glucocorticoid receptor (GR)-hsp90 heterocomplexes immunoadsorbed from L-cell cytosol, and approximately one-half of the receptors are in complexes with the FK506-binding immunophilins, and 35% with PP5. In contrast, several protein kinases (<italic> e.g</italic>. pp60src, v-Raf) exist in heterocomplexes with hsp90 and p50cdc37 . I show that the TPR proteins and p50cdc37 exist in mutually exclusive, native heterocomplexes with hsp90 and that p50cdc37 binds to a site on hsp90 that is topologically adjacent to the TPR binding site. An unresolved issue is whether there are one or two TPR binding sites per hsp90 dimer, and I show by crosslinking purified hsp90 and FKBP52 that there is a single TPR binding site on the hsp90 dimer, which explains the existence of mutually exclusive hsp90-TPR protein heterocomplexes To elucidate the mechanism for determining how v-Raf selects predominantly hsp90- p50cdc37 heterocomplexes rather than hsp90&middot;TPR protein heterocomplexes, whereas the GR selects predominantly hsp90-FKBP52 heterocomplexes and not hsp90&middot; p50cdc37 heterocomplexes, I show that p50cdc37 binds directly to Raf and that FKBP52 binds directly to the GR. I propose that a combination of exclusive binding of FKBP52 to hsp90 <italic> versus</italic> another TPR protein or p50cdc37 plus direct binding of p50cdc37 to Raf and FKBP52 to the GR allows Raf to select for the dominant Raf-hsp90&middot; p50cdc37 composition and the GR to select for the dominant GR-hsp90&middot;FKBP52 composition that is observed when they are immunoadsorbed from cytosols. Also, I show that an N-terminal fragment that contains the first two globular domain of FKBP52 competes for the binding of cytoplasmic dynein to FKBP52. This provides evidence for this N-terminal region of FKBP52 being important for its association with the molecular motor, dynein, and it may provide an attachment site for the GR heterocomplex to the same movement machinery that utilizes dynein for transporting vesicles in a retrograde direction toward the nucleus.