Different Regions of the Immunophilin FKBP52 Determine Its Association with the Glucocorticoid Receptor, hsp90, and Cytoplasmic Dynein

Abstract

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor⅐hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.

Expression profiling of glucocorticoid-treated T-ALL cell lines: rapid repression of multiple genes involved in RNA-, protein- and nucleotide synthesis

“…Readings in the shaded area constitute background+noise levels. Note that the Jurkat cell clones do not express the human GR measured by the chip, but instead high levels of stably-transfected rat GR not detected by the chip Oncogene Effects of dex treatment in lymphocytic leukemia P Obexer et al Interestingly, while the GR itself is induced by dex, three genes of the`repressed' list ( Table 2) encode proteins that have been found associated with the GR: hsp70 (Tai et al, 1992), the`immunophilin-like' protein phosphatase 5 (Silverstein et al, 1999) and a predominantly nuclear protein known under the names of glucocorticoid receptor-interacting protein (Zeiner and Gehring, 1995), receptor-associating protein of 46 kDa (RAP46; Zeiner et al, 1997) and hsp70-associated protein 46 (Hap46; Zeiner et al, 1999). RAP46 has been shown to inhibit GC-induced apoptosis (Kullmann et al, 1998).…”

“…The immunophilin moiety binds to hsp90 via its tetratricopeptide repeat domain and may be represented by any one of several proteins, such as FKBP51, FKBP52, CyP-40 or even protein phosphatase 5 (PP5) (Owens-Grillo et al, 1995;Silverstein et al, 1997Silverstein et al, , 1999. Steroid binding results in a conformational change, detachment from the associated proteins and unmasking of a nuclear translocation signal.…”

Hsp90-binding Immunophilins Link p53 to Dynein During p53 Transport to the Nucleus

“…For direct binding of purified dynamitin to purified FKBP52, myc-dynamitin was immunoadsorbed from 293-T cell cytosol and stripped of associated proteins, as described above. After washing, the immunopellets were incubated for 30 min on ice with FKBP52, purified as described previously (27), in the presence or absence of purified PPIase domain I of FKBP52. The final volume was adjusted to 50 l with HKD buffer.…”

“…GR⅐hsp90 heterocomplexes contain one of four TPR domain immunophilins: FKBP52, FKBP51, CyP-40, or PP5, the latter being a protein phosphatase that also contains TPR and PPIase homology do-mains (13,26). In these complexes, the PPIase domain functions as a protein-protein binding domain to link the GR⅐hsp90 unit to cytoplasmic dynein (27)(28)(29).…”

Independent Functions of hsp90 in Neurotransmitter Release and in the Continuous Synaptic Cycling of AMPA Receptors

“…hsp90 was proposed to be a mediator of protein trafficking over a decade ago (Pratt, 1992(Pratt, , 1993. Since then, ample evidence has accumulated indicating that hsp90 is required for the subcellular targeting of a variety of proteins, including the glucocorticoid receptor (Owens-Grillo et al, 1996;Czar et al, 1997;Silverstein et al, 1999;Galigniana et al, 2001), the dioxin receptor (Kazlauskas et al, 2001), the receptor tyrosine kinase ErbB2 (Xu et al, 2002), the epidermal growth factor receptor (Supino-Rosin et al, 2000), the CFTR (cystic fibrosis transmembrane regulator) chloride channel (Loo et al, 1998) and the G-protein G␣ 12 (Waheed and Jones, 2002). hsp90 is also required for protein translocation into mitochondria (Young et al, 2003) and peroxisomes (Crookes and Olsen, 1998).…”