Interactions of the hsp90-binding immunophilins and the dynein retrograde motor system.

Several steroid receptor-associated immunophilins, including FKBP52, CyP-40 and the protein phosphatase PPS, bind via their tetratricopeptide repeat (TPR) domains to the hsp90 component of glucocorticoid receptor (GR)•hsp90 complexes. FKBP52 has been shown to interact via its peptidylprolyl isomerase (PPIase) domain with cytoplasmic dynein, a motor protein involved in retrograde transport to the nucleus. The functional role for the PPIase domain in receptor movement was demonstrated by showing that expression of the PPIase domain fragment of FKBP52 in 3T3 cells inhibits dexamethasone-dependent nuclear translocation of the GR. This thesis discusses the protein-protein interactions that connect the hsp90-binding immunophilins to the dynein motor system, and how this interaction is evolutionarily conserved. Plants express homologues to the mammalian immunophilin FKBP52, and in wheat there are two, wFKBP73 and wFKBP77. I show that bacterially expressed wFKBPs bind to rabbit cytoplasmic dynein via their PPIase domains. In a mixed reconstitution system, the wheat hsp90/hsp70-based chaperone machinery assembles complexes containing the mouse GR, wheat hsp90, wheat FKBPs and rabbit cytoplasmic dynein. Retention by plants of the entire heterocomplex assembly machinery for linking the GR to dynein implies a fundamental role for this process in the biology of the eukaryotic cell. Here I demonstrate that, like FKBP52, both CyP-40 and PP5 interact with cytoplasmic dynein in a PPIase domain dependent manner. In 3T3 cells, PP5 is shown to colocalize with both cytoplasmic dynein and microtubules, and expression of the PPIase domain fragment of FKBP52 disrupts this localization. Also, under microtubule stabilizing conditions, tubulin is co-immunoadsorbed with the GR•hsp90 heterocomplex, showing, for the first time, a link between the GR and microtubules. Linkage of the immunophilins to cytoplasmic dynein is not direct but via the dynamitin component of the dynein-associated dynactin complex, and overexpression of dynamitin inhibits dexamethasone dependent movement of the GR in the same manner as overexpression of the PPIase domain of FKBP52. Dynamitin is recovered in native GR•hsp90 heterocomplexes when GR is immunoadsorbed from cytosol of cells expressing myc -dynamitin. These data support a model in which the immunophilins link the GR cargo through dynamitin to the dynein motor that is responsible for its retrograde movement along microtubular tracks to the nucleus.Ph.D.Health and Environmental SciencesPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124421/2/3138167.pd