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. 2009 Sep;29(17):4788-97.
doi: 10.1128/MCB.00649-09. Epub 2009 Jul 6.

Nuclear import of the glucocorticoid receptor-hsp90 complex through the nuclear pore complex is mediated by its interaction with Nup62 and importin beta

Pablo C Echeverría  1 , Gisela Mazaira, Alejandra Erlejman, Celso Gomez-Sanchez, Graciela Piwien Pilipuk, Mario D Galigniana
Affiliations

Nuclear import of the glucocorticoid receptor-hsp90 complex through the nuclear pore complex is mediated by its interaction with Nup62 and importin beta

Pablo C Echeverría et al. Mol Cell Biol. 2009 Sep.

Abstract

Glucocorticoid receptor (GR) is cytoplasmic in the absence of ligand and localizes to the nucleus after steroid binding. Previous evidence demonstrated that the hsp90-based heterocomplex bound to GR is required for the efficient retrotransport of the receptor to the nuclear compartment. We examined the putative association of GR and its associated chaperone heterocomplex with structures of the nuclear pore. We found that importin beta and the integral nuclear pore glycoprotein Nup62 interact with hsp90, hsp70, p23, and the TPR domain proteins FKBP52 and PP5. Nup62 and GR were able to interact in a more efficient manner when chaperoned by the hsp90-based heterocomplex. Interestingly, the binding of hsp70 and p23 to Nup62 does not require the presence of hsp90, whereas the association of FKBP52 and PP5 is hsp90 dependent, as indicated by the results of experiments where the hsp90 function was disrupted with radicicol. The ability of both FKBP52 and PP5 to interact with Nup62 was abrogated in cells overexpressing the TPR peptide. Importantly, GR cross-linked to the hsp90 heterocomplex was able to translocate to the nucleus in digitonin-permeabilized cells treated with steroid, suggesting that GR could pass through the pore in its untransformed state.

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Figures

FIG. 1.
FIG. 1.
In silico interactome for the GR-hsp90-TPR domain protein complex. (A) cPath-generated interactome (experimental). The query proteins are highlighted in yellow, and the importins and exportins found inside the network in red and blue, respectively. (B) Schematic of intracellular localization of the potentially associated proteins (interolog-predicted interactome). The proteins that show three or more connections with the heterocomplex are colored yellow for the GR-hsp90-TPR heterocomplex, red for importins, green for Nups, and blue for proteins of the export machinery. The proteins showing six or more connections joining the heterocomplex or going out of the heterocomplex to lower levels in the scheme are oversized.
FIG. 2.
FIG. 2.
Nup62 interacts with chaperones. (A) Coimmunoprecipitation of hsp90, hsp70, and p23 with Nups. Lane 1, whole-cell lysate from L929 fibroblasts; lane 2, nonimmune pellet; lane 3, immunoprecipitation of Nups with MAb414 antibody. (B) Nup62 is the major Nup interacting with the chaperone complex. Nup62 was immunoprecipitated with MAb414 antibody (lanes 1 and 3) or a nonimmune antibody (lane 2). The pellet was stripped of associated proteins (lanes 2 and 3), and the Nup-chaperone complex was reconstituted with reticulocyte lysate in the presence of an ATP-regenerating system. (C) Binding of hsp70 and p23 to Nup62 is independent of hsp90. Lanes 1 to 3, Nup62 was immunoprecipitated, stripped, and reconstituted with reticulocyte lysate. Lane 1, nonimmune pellet; lane 2, complex reconstituted with reticulocyte lysate; lane 3, complex reconstituted in the presence of 2 μM radicicol. Lanes 4 to 6, Nup62 was immunoprecipitated from L929 cells. Lane 4, nonimmune pellet; lane 5, MAb414 immune pellet; lane 6, immune pellet from cells pretreated with 2 μM radicicol for 3 h.
FIG. 3.
FIG. 3.
GR and hsp90 bind to Impβ. (A) Nup62 and Impβ1 coimmunoprecipitate with hsp90. Lane 1, L929 cell lysate; lane 2, nonimmune pellet; lane 3, immunoprecipitation of hsp90. (B) Impβ1 coimmunoprecipitates with GR. Lane 1, L929 cell lysate; lane 2, nonimmune pellet; lane 3, immunoprecipitation of GR. (C) Knockdown of Impβ with specific siRNA in HEK293T cell lysates. (D) Nuclear translocation rate of GFP-GR in normal HEK293T cells (○) and cells treated with the siRNA against Impβ (•). Receptor translocation was triggered with 100 nM dexamethasone added to the medium at zero time. Results are the means ± standard errors of the means from three independent experiments where ∼100 cells were counted for each time point. *, significantly different at P < 0.005.
FIG. 4.
FIG. 4.
GR interacts with Nup62. Top left, coimmunoprecipitation of Nup62 with GR. Lane 1, L929 cell lysate; lane 2, nonimmune pellet; lane 3, immunoprecipitation of GR. Top right and bottom, confocal microscopy. L929 cells or the L929-derived GR−/− cell line E82.A3 were treated with 10 nM cortisol for 12 min, fixed with cold methanol, and stained for endogenous GR with BuGR2 antibody and for Nups with MAb414 antibody (counterstained with Alexa 488-labeled and rhodamine-labeled secondary antibody, respectively). GR (green signal) overlapping with the perinuclear ring (red signal) obtained with the MAb414 anti-Nup antibody shows a yellow signal (dashed boxes). This yellow merged image was not observed without steroid or after 20 min with steroid (data not shown).
FIG. 5.
FIG. 5.
GR interacts with Nup62 directly. (A) Nup62 was immunoprecipitated, stripped, and reincubated with pure GST (lane 1) or pure GST-GR (lane 2), or it was incubated with reticulocyte lysate, washed, and then treated with pure GST-GR (lane 3); Nup62 pellet treated as described for lane 3 was incubated with the GST-GR heterocomplex previously reconstituted with reticulocyte lysate (lane 4); Nup62 pellet treated as described for lane 4 was incubated in the presence of 2 μM radicicol (lane 5); and nonimmune Nup62 pellet was incubated with pure GST-GR (lane 6). GST-GR was revealed with an anti-GST antibody. (B) Effects of soluble cytosolic factors and GTP on GR-Nup62 binding. Nup62 immunopellets were reconstituted with reticulocyte lysate and incubated with GST-GR-hsp90 heterocomplex reconstituted as described for lane 4 of panel A (lane 1), 50 μM GTP (lane 2), 40 μg of cytosolic proteins from E82.A3 lysates (lane 3), or cytosolic proteins and GTP combined (lane 4), and nonimmune Nup62 pellet was incubated with pure GST-GR (lane 5). “Input” represents one-fifth of the total amount of GST (lane 1) or GST-GR (lanes 2 to 6) used in the incubation with the Nup62 immunopellet.
FIG. 6.
FIG. 6.
Nup62 binds hsp90-binding TPR proteins. (A) L929 cells were incubated on ice with ethanol (−) or dexamethasone (+). After 1 h of incubation, GR was immunoprecipitated and the associated proteins were visualized by Western blotting. NI, nonimmune antibody. (B) TPR protein association is hsp90 dependent. Nup62 was immunoprecipitated with MAb414 antibody (lanes 2 to 4) or nonimmune antibody (lane 1) and incubated with buffer (lane 2), reticulocyte lysate (lanes 1 and 3), or reticulocyte lysate and 2 μM radicicol (lane 4). (C) IMMs bind to Nup62 via their TPR domains. Stripped Nup62 immunopellets were incubated with reticulocyte lysate supplemented with buffer (lane 1), TPR peptide (lane 2), or peptidyl-prolyl isomerase peptide (lane 3). (D) Confocal microscopy following indirect immunofluorescence assay for Nup62 (red) and the indicated IMMs (green). “TPR” indicates cells overexpressing the TPR peptide. The yellow in the merged images shows colocalization of Nup62 with FKBP2 and was impaired by overexpression of the TPR domain of PP5.
FIG. 7.
FIG. 7.
The GR-hsp90-based heterocomplex passes intact through the NPC. Cytosolic mouse GR (mGR) produced by baculovirus infection of Sf9 cells was cross-linked to the insect chaperone heterocomplex with 2 mM DSP. The Western blot shows the association of insect hsp90 and hsp70 with GR in untreated cytosol (lane 1), cytosol heated in the presence of an excess of β-mercaptoethanol (lane 2), or sample buffer without β-mercaptoethanol (lane 3). Confocal microscopy was performed on permeabilized E82.A3 cells incubated with GR cross-linked with DSP in Sf9 cytosol in the absence or presence of 100 nM dexamethasone. GR is seen as green, and the perinuclear ring of Nups is seen as red. Controls incubated without ATP and cytosol showed GR, with steroid, entirely cytoplasmic (data not shown).
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