Limits...
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.

Show MeSH

Related in: MedlinePlus

Hormone-bound  nuclear GR in Hypo buffer– extracted cells is associated  with chromatin. GrH2 cells  grown on coverslips were  treated with 1 μM of corticosterone for 1 h. After  permeabilization with digitonin, nuclei were either extracted with Hypo buffer (A  and B), or extracted and then  incubated with 100 μg/ml of  RNase-free DNase I for 30  min at room temperature  (C–F). GR (A, C, and E) and  DNA (B, D, and F) in fixed  cells were visualized by IIF  using BuGR2 and DAPI  staining, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139874&req=5

Figure 2: Hormone-bound nuclear GR in Hypo buffer– extracted cells is associated with chromatin. GrH2 cells grown on coverslips were treated with 1 μM of corticosterone for 1 h. After permeabilization with digitonin, nuclei were either extracted with Hypo buffer (A and B), or extracted and then incubated with 100 μg/ml of RNase-free DNase I for 30 min at room temperature (C–F). GR (A, C, and E) and DNA (B, D, and F) in fixed cells were visualized by IIF using BuGR2 and DAPI staining, respectively.

Mentions: Hormone-bound GRs are known to associate with chromatin upon their import into nuclei (Yamamoto and Alberts, 1976). To confirm that hormone-bound nuclear GRs in Hypo buffer–extracted cells were chromatin bound, hormone-treated, permeabilized cells were subjected to a DNase I digestion after the Hypo buffer extraction. In agreement with our previous results, GRs in hormone-treated, permeabilized cells were resistant to Hypo buffer extraction (Fig. 2 A). However, treatment of Hypo buffer–extracted cells with DNase I led to the loss of nearly all GRs from most nuclei (Fig. 2 E). If the extent of DNase I digestion was varied, the relative amount of hormone-bound GR that remained nuclear in Hypo buffer–extracted cells was correlated with the relative amount of DNA that remained within nuclei (Fig. 2, C and D). The nuclear matrix protein NuMA was not significantly extracted by either Hypo buffer or DNase I digestion (not shown), indicating that the nuclear matrix remained intact under these conditions. Thus, since chromatin-bound receptors in hormonetreated cells are predominantly resistant to hypotonic extraction, it appears likely that the sensitivity of GRs to Hypo buffer extraction in hormone-withdrawn cells is due to their rapid release from chromatin.


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

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

Hormone-bound  nuclear GR in Hypo buffer– extracted cells is associated  with chromatin. GrH2 cells  grown on coverslips were  treated with 1 μM of corticosterone for 1 h. After  permeabilization with digitonin, nuclei were either extracted with Hypo buffer (A  and B), or extracted and then  incubated with 100 μg/ml of  RNase-free DNase I for 30  min at room temperature  (C–F). GR (A, C, and E) and  DNA (B, D, and F) in fixed  cells were visualized by IIF  using BuGR2 and DAPI  staining, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139874&req=5

Figure 2: Hormone-bound nuclear GR in Hypo buffer– extracted cells is associated with chromatin. GrH2 cells grown on coverslips were treated with 1 μM of corticosterone for 1 h. After permeabilization with digitonin, nuclei were either extracted with Hypo buffer (A and B), or extracted and then incubated with 100 μg/ml of RNase-free DNase I for 30 min at room temperature (C–F). GR (A, C, and E) and DNA (B, D, and F) in fixed cells were visualized by IIF using BuGR2 and DAPI staining, respectively.
Mentions: Hormone-bound GRs are known to associate with chromatin upon their import into nuclei (Yamamoto and Alberts, 1976). To confirm that hormone-bound nuclear GRs in Hypo buffer–extracted cells were chromatin bound, hormone-treated, permeabilized cells were subjected to a DNase I digestion after the Hypo buffer extraction. In agreement with our previous results, GRs in hormone-treated, permeabilized cells were resistant to Hypo buffer extraction (Fig. 2 A). However, treatment of Hypo buffer–extracted cells with DNase I led to the loss of nearly all GRs from most nuclei (Fig. 2 E). If the extent of DNase I digestion was varied, the relative amount of hormone-bound GR that remained nuclear in Hypo buffer–extracted cells was correlated with the relative amount of DNA that remained within nuclei (Fig. 2, C and D). The nuclear matrix protein NuMA was not significantly extracted by either Hypo buffer or DNase I digestion (not shown), indicating that the nuclear matrix remained intact under these conditions. Thus, since chromatin-bound receptors in hormonetreated cells are predominantly resistant to hypotonic extraction, it appears likely that the sensitivity of GRs to Hypo buffer extraction in hormone-withdrawn cells is due to their rapid release 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