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Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export.

Yang J, Liu J, DeFranco DB - J. Cell Biol. (1997)

Bottom Line: Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality.If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited.Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

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(a) Western blot analysis of molybdate-stimulated in  vitro GR nuclear export. (Lanes 1–8) GrH2 cells treated with  corticosterone (Cort) for 1 h and then withdrawn from hormone  for 20 min. (Lanes 9–12) GrH2 cells treated with corticosterone  for 80 min. Harvested cells were permeabilized in suspension,  and aliquots of intact nuclei were incubated with 50 μl of reaction  mixture containing 10 mg/ml BSA in transport buffer, 4 mM ATP  (lanes 1, 2, 5, 6, 9, and 10), or 4 mM GTP (lanes 3, 7, and 11) with  an energy-regenerating system, and 20 mM sodium molybdate  where indicated (lanes 2, 3, 4, 6, 7, 8, 10, 11, and 12). After a 20min incubation at 30°C, each nuclear suspension was split into  two identical samples. One sample was subjected to SDS-PAGE  directly (lanes 1–4, Whole). The other identical sample was  washed, and nuclei were recovered and subjected to SDS-PAGE  for detection of the remaining nuclear GR (lanes 5–12, Nuc  Prep). (b) Quantification of GR levels observed in Western blots  by densitometry. GR levels were normalized to the internal control NuMA protein. (Bars 1–8) Average of four experiments;  (bars 9–12) average of two experiments. Whole, whole reaction  mix; Nuc Prep, nuclear pellets.
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Figure 7: (a) Western blot analysis of molybdate-stimulated in vitro GR nuclear export. (Lanes 1–8) GrH2 cells treated with corticosterone (Cort) for 1 h and then withdrawn from hormone for 20 min. (Lanes 9–12) GrH2 cells treated with corticosterone for 80 min. Harvested cells were permeabilized in suspension, and aliquots of intact nuclei were incubated with 50 μl of reaction mixture containing 10 mg/ml BSA in transport buffer, 4 mM ATP (lanes 1, 2, 5, 6, 9, and 10), or 4 mM GTP (lanes 3, 7, and 11) with an energy-regenerating system, and 20 mM sodium molybdate where indicated (lanes 2, 3, 4, 6, 7, 8, 10, 11, and 12). After a 20min incubation at 30°C, each nuclear suspension was split into two identical samples. One sample was subjected to SDS-PAGE directly (lanes 1–4, Whole). The other identical sample was washed, and nuclei were recovered and subjected to SDS-PAGE for detection of the remaining nuclear GR (lanes 5–12, Nuc Prep). (b) Quantification of GR levels observed in Western blots by densitometry. GR levels were normalized to the internal control NuMA protein. (Bars 1–8) Average of four experiments; (bars 9–12) average of two experiments. Whole, whole reaction mix; Nuc Prep, nuclear pellets.

Mentions: The results of the IIF assay were confirmed by Western blots, which analyzed GR nuclear export from permeabilized cells maintained in suspension. This assay provided a more quantitative assessment of in vitro GR nuclear export that was particularly useful for dose–response analysis of the various compounds tested (Table I). GrH2 cells were treated with hormone for 1 h, and then either withdrawn from hormone for 20 min (Fig. 7, lanes 1–8) or maintained in hormone-containing medium for an additional 20 min (Fig. 7, lanes 9–12). After permeabilization, intact nuclei were incubated with BSA at 30°C for 20 min, with or without sodium molybdate and/or ATP. Nuclear suspensions were split into two identical samples after the in vitro incubation. SDS sample buffer was added to one sample, which was immediately subjected to SDS-PAGE and Western blot analysis to reveal overall GR levels and the integrity of the receptor (Fig. 7, lanes 1–4). The other sample was washed, and GR remaining within nuclei was visualized by Western blot analysis (Fig. 7, lanes 5–12). Similar amounts of intact GR were recovered under all conditions (Fig. 7, lanes 1–4), indicating that the reduction in nuclear GR levels that occurred upon sodium molybdate treatment (Fig. 7, lanes 6 and 7) resulted from active GR nuclear export and not degradation. 20 mM sodium molybdate was chosen for subsequent assays since it was effective in the stimulation of GR nuclear export (Table I) and did not generate abnormal nuclear morphology sometimes associated with higher doses. In the suspension assay shown in Fig. 7, sodium molybdate treatment in the presence of ATP led to the export of ∼80% of nuclear GR (lane 6) while, in the presence of GTP, ∼40% of nuclear GR was exported (lane 7). Molybdate alone did not induce GR export in the absence of ATP or GTP (Fig. 7, lane 8), or in the presence of nonhydrolyzable ATP or GTP analogs (i.e., ATPγS, AMP-PNP, or GTPγS, respectively, not shown). Finally, in cells that were not withdrawn from hormone so that GRs remained tightly bound to chromatin (Fig. 7, lanes 9–12), molybdate exerted a limited effect on GR nuclear export. Thus, energy-dependent, in vitro nuclear export of GRs is most effective when receptors are released from chromatin.


Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export.

Yang J, Liu J, DeFranco DB - J. Cell Biol. (1997)

(a) Western blot analysis of molybdate-stimulated in  vitro GR nuclear export. (Lanes 1–8) GrH2 cells treated with  corticosterone (Cort) for 1 h and then withdrawn from hormone  for 20 min. (Lanes 9–12) GrH2 cells treated with corticosterone  for 80 min. Harvested cells were permeabilized in suspension,  and aliquots of intact nuclei were incubated with 50 μl of reaction  mixture containing 10 mg/ml BSA in transport buffer, 4 mM ATP  (lanes 1, 2, 5, 6, 9, and 10), or 4 mM GTP (lanes 3, 7, and 11) with  an energy-regenerating system, and 20 mM sodium molybdate  where indicated (lanes 2, 3, 4, 6, 7, 8, 10, 11, and 12). After a 20min incubation at 30°C, each nuclear suspension was split into  two identical samples. One sample was subjected to SDS-PAGE  directly (lanes 1–4, Whole). The other identical sample was  washed, and nuclei were recovered and subjected to SDS-PAGE  for detection of the remaining nuclear GR (lanes 5–12, Nuc  Prep). (b) Quantification of GR levels observed in Western blots  by densitometry. GR levels were normalized to the internal control NuMA protein. (Bars 1–8) Average of four experiments;  (bars 9–12) average of two experiments. Whole, whole reaction  mix; Nuc Prep, nuclear pellets.
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Figure 7: (a) Western blot analysis of molybdate-stimulated in vitro GR nuclear export. (Lanes 1–8) GrH2 cells treated with corticosterone (Cort) for 1 h and then withdrawn from hormone for 20 min. (Lanes 9–12) GrH2 cells treated with corticosterone for 80 min. Harvested cells were permeabilized in suspension, and aliquots of intact nuclei were incubated with 50 μl of reaction mixture containing 10 mg/ml BSA in transport buffer, 4 mM ATP (lanes 1, 2, 5, 6, 9, and 10), or 4 mM GTP (lanes 3, 7, and 11) with an energy-regenerating system, and 20 mM sodium molybdate where indicated (lanes 2, 3, 4, 6, 7, 8, 10, 11, and 12). After a 20min incubation at 30°C, each nuclear suspension was split into two identical samples. One sample was subjected to SDS-PAGE directly (lanes 1–4, Whole). The other identical sample was washed, and nuclei were recovered and subjected to SDS-PAGE for detection of the remaining nuclear GR (lanes 5–12, Nuc Prep). (b) Quantification of GR levels observed in Western blots by densitometry. GR levels were normalized to the internal control NuMA protein. (Bars 1–8) Average of four experiments; (bars 9–12) average of two experiments. Whole, whole reaction mix; Nuc Prep, nuclear pellets.
Mentions: The results of the IIF assay were confirmed by Western blots, which analyzed GR nuclear export from permeabilized cells maintained in suspension. This assay provided a more quantitative assessment of in vitro GR nuclear export that was particularly useful for dose–response analysis of the various compounds tested (Table I). GrH2 cells were treated with hormone for 1 h, and then either withdrawn from hormone for 20 min (Fig. 7, lanes 1–8) or maintained in hormone-containing medium for an additional 20 min (Fig. 7, lanes 9–12). After permeabilization, intact nuclei were incubated with BSA at 30°C for 20 min, with or without sodium molybdate and/or ATP. Nuclear suspensions were split into two identical samples after the in vitro incubation. SDS sample buffer was added to one sample, which was immediately subjected to SDS-PAGE and Western blot analysis to reveal overall GR levels and the integrity of the receptor (Fig. 7, lanes 1–4). The other sample was washed, and GR remaining within nuclei was visualized by Western blot analysis (Fig. 7, lanes 5–12). Similar amounts of intact GR were recovered under all conditions (Fig. 7, lanes 1–4), indicating that the reduction in nuclear GR levels that occurred upon sodium molybdate treatment (Fig. 7, lanes 6 and 7) resulted from active GR nuclear export and not degradation. 20 mM sodium molybdate was chosen for subsequent assays since it was effective in the stimulation of GR nuclear export (Table I) and did not generate abnormal nuclear morphology sometimes associated with higher doses. In the suspension assay shown in Fig. 7, sodium molybdate treatment in the presence of ATP led to the export of ∼80% of nuclear GR (lane 6) while, in the presence of GTP, ∼40% of nuclear GR was exported (lane 7). Molybdate alone did not induce GR export in the absence of ATP or GTP (Fig. 7, lane 8), or in the presence of nonhydrolyzable ATP or GTP analogs (i.e., ATPγS, AMP-PNP, or GTPγS, respectively, not shown). Finally, in cells that were not withdrawn from hormone so that GRs remained tightly bound to chromatin (Fig. 7, lanes 9–12), molybdate exerted a limited effect on GR nuclear export. Thus, energy-dependent, in vitro nuclear export of GRs is most effective when receptors are released from chromatin.

Bottom Line: Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality.If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited.Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

Show MeSH
Related in: MedlinePlus