The hsp56 immunophilin component of untransformed steroid receptor complexes is localized both to microtubules in the cytoplasm and to the same nonrandom regions within the nucleus as the steroid receptor.

223

232

Here, we also show that native GR⅐hsp90 heterocomplexes immunoadsorbed from L cell cytosol contain dynein and that GR⅐hsp90 heterocomplexes assembled in reticulocyte lysate contain cytoplasmic dynein in a manner that is competed by the PPIase domain of FKBP52.Steroid receptors move continuously into and out of the nucleus (Refs. 1-4; for review, see Ref. 5), and, depending upon the receptor, the hormone-free, untransformed receptor may have a predominantly nuclear or cytoplasmic localization. The hormone-free glucocorticoid receptor (GR) 1 is localized to the cytoplasm of most cells, and after steroid binding and transformation, it translocates to the nucleus (6 -8). Several studies with inhibitors suggest that the multiprotein hsp90-based chaperone system and the hsp90-binding immunophilin FKBP52 are involved in movement of the GR along microtubular tracks to the nucleus (for review, see Ref. 9). Assembly of receptors into heterocomplexes with hsp90 is a dynamic process (10), and it has been shown that the GR and hsp90 can move together from the cytoplasm to the nucleus (11). A couple of observations suggest that the role of hsp90 in receptor movement is likely to involve dynamic assembly and disassembly of GR⅐hsp90 heterocomplexes. For example, Yang and DeFranco (12) showed that molybdate, which binds to hsp90 and stabilizes GR⅐hsp90 heterocomplexes in vivo (13), traps the GR in the cytoplasm of cells continuously exposed to hormone. Molybdate in this case was thought to inhibit reimport of the GR into the nucleus by inhibiting the dynamic cycling of receptors into and out of their complexes with the hsp90 chaperone. Also, geldanamycin, an antibiotic that binds to the nucleotide binding site on hsp90 (14) and prevents formation of normal receptor⅐hsp90 heterocomplexes (15), impedes steroid-induced movement of the GR from the cytoplasm to the nucleus (16, 17).Some localization studies have shown the untransformed GR to colocalize with microtubules (for review, see Ref. 18), but the evidence supporting movement along microtubular tracks is indirect. Although microtubule disrupting agents, such as colcemid, do not affect the overall rate of steroid-dependent receptor translocation to the nucleus (8, 19), they eliminate the hsp90-dependent mode of receptor movement (17). Using a fusion protein of murine GR with Aequorea green fluorescent protein (GFP), we found that steroid-dependent GFP-GR translocation to the nucleus is rapid (t1 ⁄2 ϭ ϳ5 min) both in cells with intact cytoskeleton and in cells with disrupted cytoskeletal networks (17). However, in cells with normal cytoskeleton, the hsp90 inhibitor geldanamycin slowed translocation of the GFP-GR by an order of magnitude (t1 ⁄2 ϭ ϳ45 min), whereas in cells with colcemid-disrupted microtubules, geldanamycin had no effect on the translocation rate (t1 ⁄2 ϭ ϳ5 min). This suggests two mechanisms of GR movement. Under physiological conditions where the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery in which the hsp90 heter...

supporting

confidence: 81%

“…5 for NL1 composition) is sufficient for FKBP52 binding (28). FKBP52 binds either directly or via a linker protein with cytoplasmic dynein (29), and coimmunoadsorption of dynein is competed by a fragment of FKBP52 comprising its PPIase domain (28).…”

mentioning

confidence: 99%

Evidence That the Peptidylprolyl Isomerase Domain of the hsp90-binding Immunophilin FKBP52 Is Involved in Both Dynein Interaction and Glucocorticoid Receptor Movement to the Nucleus

Galigniana

Radanyi

Renoir

et al. 2001

223

232

“…Interestingly, the GR receptor heterocomplexes of WCL2 cells (Fig. 1B) are localized to the nucleus where they are distributed in a mottled, nonrandom fashion and are excluded from nucleoli (59), and we have shown by double immunofluorescence and confocal imaging that the GR and FKBP52 are co-localized within the WCL2 cell nucleus (58). Thus, the localization of both FKBP52 and PP5 is consistent with a potential role in nuclear trafficking of receptors.…”

Section: Fig 6 a Potential Fk506 Binding Domain In Pp5supporting

confidence: 53%

Protein Phosphatase 5 Is a Major Component of Glucocorticoid Receptor·hsp90 Complexes with Properties of an FK506-binding Immunophilin

Silverstein

Galigniana

Chen

et al. 1997

242

208

Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506-or cyclosporin Abinding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315-32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormonefree cells. Approximately one-half of the GR⅐hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and ϳ35% contains PP5. Only a small (but undetermined) fraction of the native GR⅐hsp90 heterocomplexes contain the cyclosporin Abinding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [ 3 H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol.In cytosols prepared from hormone-free cells, steroid receptors exist in multiprotein complexes that contain hsp90 1 and some hsp90-associated proteins, including p23 and some high molecular weight immunophilins (for review see Refs. 1 and 2). The immunophilins are ubiquitous and conserved proteins that bind immunosuppressant drugs, such as FK506 and cyclosporin A (for review see Ref.3). All members of the immunophilin family have peptidylprolyl isomerase (PPIase) activity, and there are two classes: the FKBPs that bind compounds like FK506 and rapamycin and the cyclophilins (CyPs) that bind cyclosporin A. The drugs bind to the isomerase site on the immunophilin and inhibit cis-trans isomerization in vitro (4).The low molecular weight immunophilins, such as FKBP12 and CyP-18, are thought to be the cellular components responsible for the immunosuppression and are the most studied. Three high molecular weight immunophilins, FK...

“…In this respect, STAT5B was already shown to interact directly with the glucocorticoid receptor (GR) in hepatic and mammary cells (34,35). Interestingly, the GR is also MT-bound (3) and is associated with hsp90 (36,37), the FKBP52 immunophilin (38) and the dynein intermediate chain (39). Whether STAT5B would participate in such complexes and whether these would actually favor its interaction with other signaling molecules remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

STAT5B-mediated Growth Hormone Signaling Is Organized by Highly Dynamic Microtubules in Hepatic Cells

Phung-Koskas

Pilon

Poüs

et al. 2005

In the last decade, the notion that microtubules are critical to the spatial organization of signal transduction and contribute to the transmission of signals to downstream targets has been proposed. Because the STAT5B transduction and transcription factor is the major STAT protein activated by growth hormone stimulation in hepatocytes and is a crossroads between many signaling pathways, we studied the involvement of microtubules in STAT5B-mediated growth hormone signaling pathway in the highly differentiated and polarized WIF-B hepatic cell line. We showed that depolymerization of the microtubule network impaired STAT5B translocation to the nucleus upon growth hormone treatment. A significant amount of STAT5B binds to microtubules, while STAT5A and STAT3 are exclusively compartmentalized in the cytosol. Moreover, taxol-induced stabilization of microtubules released STAT5B from its binding, and we show that STAT5B binds specifically to the highly dynamic microtubules and is absent of the stable microtubule subpopulation. The specific involvement of dynamic microtubule subpopulation in growth hormone signaling pathway was confirmed by the inhibition of growth hormone-induced STAT5B nuclear translocation after stabilization of microtubules or specific disruption of highly dynamic microtubules. Upon growth hormone treatment, MT-bound STAT5B was rapidly released from microtubules by a dynein-dependent transport to the nucleus. Altogether, our findings indicate that the labile microtubule subpopulation specifically and dynamically organizes STAT5B-mediated growth hormone signaling in hepatic cells.Once bound to specific receptors, a large number of cytokines, growth factors, and hormones act through a sequence of steps allowing the rapid transport of information from the plasma membrane to subcellular targets. This information often results in the phosphorylation of transcription factors that subsequently migrate to the nucleus where they initiate specific gene transcription. How transcription factors are organized as molecular targets downstream of receptor activation and how they move rapidly through the cytoplasm from their activation site to the nucleus often remains unclear. The notion that microtubules (MTs) 1 might be involved in the cytoplasmic activation and/or trafficking of various transcription factors and protein kinases has emerged, as many signal transduction molecules, including transcription factors and protein kinases, have been shown to interact with MTs (1). For example, the glucocorticoid receptor was shown to colocalize with the MT network in mammalian cells (2, 3). The tumor suppressor protein p53 also uses MT tracks to migrate to the nucleus (4). Various protein kinases such as the MAP kinase kinase kinase MLK2 (5) or the MAP kinases ERK1 and ERK2 (6) were also shown to interact or to colocalize with MTs or with microtubule-associated proteins (7).In this study, we investigated the putative involvement of the MT network in GH signaling in the highly differentiated WIF-B hepatic cell line (8, 9...