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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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Genistein blocks molybdate stimulation of protein tyrosine phosphorylation and in vitro nuclear export of GR. Hormone-withdrawn GrH2 cells were permeabilized in suspension,  and recovered nuclei were incubated with the following components: 4 mM ATP (lane 1); 4 mM ATP and 20 mM sodium molybdate (lane 2); ATP, molybdate, and 0.2 mM genistein (lane 3);  and molybdate (lane 4). After a 20-min incubation at 30°C, nuclei  were recovered and nuclear proteins were subjected to SDSPAGE. Half of the sample was subjected to Western blot analysis  to detect phosphotyrosine (p-Tyr) using the PY20 anti-phosphotyrosine mAb, while the other half of the sample was subjected to  Western blot analysis to detect GR and NuMA.
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Figure 12: Genistein blocks molybdate stimulation of protein tyrosine phosphorylation and in vitro nuclear export of GR. Hormone-withdrawn GrH2 cells were permeabilized in suspension, and recovered nuclei were incubated with the following components: 4 mM ATP (lane 1); 4 mM ATP and 20 mM sodium molybdate (lane 2); ATP, molybdate, and 0.2 mM genistein (lane 3); and molybdate (lane 4). After a 20-min incubation at 30°C, nuclei were recovered and nuclear proteins were subjected to SDSPAGE. Half of the sample was subjected to Western blot analysis to detect phosphotyrosine (p-Tyr) using the PY20 anti-phosphotyrosine mAb, while the other half of the sample was subjected to Western blot analysis to detect GR and NuMA.

Mentions: To confirm that genistein blocked molybdate-stimulated protein tyrosine phosphorylation, we performed Western blots with an anti-phosphotyrosine antibody. These blots were also costained with GR and NuMA to confirm GR nuclear export visualized by IIF in Fig. 11. As shown in Fig. 12, relatively low levels of protein tyrosine phosphorylation were observed in permeabilized cells treated with either ATP (Fig. 12, lane 1) or sodium molybdate alone (Fig. 12, lane 4). Under these conditions, GR is effectively retained within nuclei. Treatment of permeabilized cells with ATP and molybdate dramatically increased the overall level of protein tyrosine phosphorylation, and correspondingly stimulated nuclear export of GR (Fig. 12, lane 2). Thus, increased tyrosine phosphorylation induced by molybdate corresponds with the stimulation of GR nuclear export. In agreement with our IIF data (Fig. 11), genistein blocked molybdate-stimulated GR nuclear export (Fig. 12, middle). In addition, genistein also decreased the extent of molybdate-induced protein tyrosine phosphorylation in permeabilized cells (Fig. 12, lane 3). Thus, our results are consistent with the possible involvement of some protein phosphotyrosine system in the nuclear export of GR and perhaps other shuttling proteins.


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

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

Genistein blocks molybdate stimulation of protein tyrosine phosphorylation and in vitro nuclear export of GR. Hormone-withdrawn GrH2 cells were permeabilized in suspension,  and recovered nuclei were incubated with the following components: 4 mM ATP (lane 1); 4 mM ATP and 20 mM sodium molybdate (lane 2); ATP, molybdate, and 0.2 mM genistein (lane 3);  and molybdate (lane 4). After a 20-min incubation at 30°C, nuclei  were recovered and nuclear proteins were subjected to SDSPAGE. Half of the sample was subjected to Western blot analysis  to detect phosphotyrosine (p-Tyr) using the PY20 anti-phosphotyrosine mAb, while the other half of the sample was subjected to  Western blot analysis to detect GR and NuMA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139874&req=5

Figure 12: Genistein blocks molybdate stimulation of protein tyrosine phosphorylation and in vitro nuclear export of GR. Hormone-withdrawn GrH2 cells were permeabilized in suspension, and recovered nuclei were incubated with the following components: 4 mM ATP (lane 1); 4 mM ATP and 20 mM sodium molybdate (lane 2); ATP, molybdate, and 0.2 mM genistein (lane 3); and molybdate (lane 4). After a 20-min incubation at 30°C, nuclei were recovered and nuclear proteins were subjected to SDSPAGE. Half of the sample was subjected to Western blot analysis to detect phosphotyrosine (p-Tyr) using the PY20 anti-phosphotyrosine mAb, while the other half of the sample was subjected to Western blot analysis to detect GR and NuMA.
Mentions: To confirm that genistein blocked molybdate-stimulated protein tyrosine phosphorylation, we performed Western blots with an anti-phosphotyrosine antibody. These blots were also costained with GR and NuMA to confirm GR nuclear export visualized by IIF in Fig. 11. As shown in Fig. 12, relatively low levels of protein tyrosine phosphorylation were observed in permeabilized cells treated with either ATP (Fig. 12, lane 1) or sodium molybdate alone (Fig. 12, lane 4). Under these conditions, GR is effectively retained within nuclei. Treatment of permeabilized cells with ATP and molybdate dramatically increased the overall level of protein tyrosine phosphorylation, and correspondingly stimulated nuclear export of GR (Fig. 12, lane 2). Thus, increased tyrosine phosphorylation induced by molybdate corresponds with the stimulation of GR nuclear export. In agreement with our IIF data (Fig. 11), genistein blocked molybdate-stimulated GR nuclear export (Fig. 12, middle). In addition, genistein also decreased the extent of molybdate-induced protein tyrosine phosphorylation in permeabilized cells (Fig. 12, lane 3). Thus, our results are consistent with the possible involvement of some protein phosphotyrosine system in the nuclear export of GR and perhaps other shuttling proteins.

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