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

Selective effects  of tungstate on in vitro nuclear protein export. Permeabilized HeLa cells were incubated for 20 min at 30°C  with BSA in transport buffer  containing 20 mM sodium  tungstate, in the absence (A,  C, E, and G) or presence of 4  mM ATP (B, D, F, and H).  Fixed cells were subjected to  costaining to detect either  hnRNP A1 (A and B) and  hsp56 (C and D), or hnRNP  C (G and H) and hsp90 (E  and F) in the same fields of  cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139874&req=5

Figure 10: Selective effects of tungstate on in vitro nuclear protein export. Permeabilized HeLa cells were incubated for 20 min at 30°C with BSA in transport buffer containing 20 mM sodium tungstate, in the absence (A, C, E, and G) or presence of 4 mM ATP (B, D, F, and H). Fixed cells were subjected to costaining to detect either hnRNP A1 (A and B) and hsp56 (C and D), or hnRNP C (G and H) and hsp90 (E and F) in the same fields of cells.

Mentions: Since nuclear export of unliganded GR and that of hsp90 appear to be independent events which are both triggered by sodium molybdate, we expanded our analysis to include hsp56, another GR associated protein, and distinct members of the heterogeneous ribonuclear protein family that differ in their nucleocytoplasmic shuttling properties (i.e., hnRNP A1 and hnRNP C) (Pinol-Roma and Dreyfuss, 1992). In our assessment of in vitro nuclear export of hnRNP A1, hnRNP C, and hsp56, we used tungstate, a group VI-A transition metal oxyanion that was found to be more effective than molybdate. As shown in Fig. 10, in the absence of ATP, all four proteins were retained within nuclei of permeabilized cells after a 20-min incubation with sodium tungstate (Fig. 10, A, C, E, and G). However, in the presence of ATP, hnRNP A1 (Fig. 10 B) and hsp 90 (Fig. 10 F) efficiently exported from the nuclei after incubation with sodium tungstate while, in the same field of the cells, hsp56 (Fig. 10 D) and hnRNP C (Fig. 10 H), respectively, were retained within nuclei. Tungstate also induced rapid in vitro nuclear export of hsp70 (not shown). hsp90 is distributed in both the cytoplasm and nucleus (Gasc et al., 1990), indicating that it might be a shuttling protein, although this has not been experimentally verified. hsp56 is found predominantly within the nucleus (Czar et al., 1995) and its nucleocytoplasmic shuttling properties have not been assessed. The fact that molybdate and tungstate effects are exerted on hsp70, a shuttling protein, and a shuttling hnRNP protein (i.e., hnRNP A1) suggests that some component of the nuclear export machinery used by shuttling proteins may be targeted by metal oxyanions.


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

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

Selective effects  of tungstate on in vitro nuclear protein export. Permeabilized HeLa cells were incubated for 20 min at 30°C  with BSA in transport buffer  containing 20 mM sodium  tungstate, in the absence (A,  C, E, and G) or presence of 4  mM ATP (B, D, F, and H).  Fixed cells were subjected to  costaining to detect either  hnRNP A1 (A and B) and  hsp56 (C and D), or hnRNP  C (G and H) and hsp90 (E  and F) in the same fields of  cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Selective effects of tungstate on in vitro nuclear protein export. Permeabilized HeLa cells were incubated for 20 min at 30°C with BSA in transport buffer containing 20 mM sodium tungstate, in the absence (A, C, E, and G) or presence of 4 mM ATP (B, D, F, and H). Fixed cells were subjected to costaining to detect either hnRNP A1 (A and B) and hsp56 (C and D), or hnRNP C (G and H) and hsp90 (E and F) in the same fields of cells.
Mentions: Since nuclear export of unliganded GR and that of hsp90 appear to be independent events which are both triggered by sodium molybdate, we expanded our analysis to include hsp56, another GR associated protein, and distinct members of the heterogeneous ribonuclear protein family that differ in their nucleocytoplasmic shuttling properties (i.e., hnRNP A1 and hnRNP C) (Pinol-Roma and Dreyfuss, 1992). In our assessment of in vitro nuclear export of hnRNP A1, hnRNP C, and hsp56, we used tungstate, a group VI-A transition metal oxyanion that was found to be more effective than molybdate. As shown in Fig. 10, in the absence of ATP, all four proteins were retained within nuclei of permeabilized cells after a 20-min incubation with sodium tungstate (Fig. 10, A, C, E, and G). However, in the presence of ATP, hnRNP A1 (Fig. 10 B) and hsp 90 (Fig. 10 F) efficiently exported from the nuclei after incubation with sodium tungstate while, in the same field of the cells, hsp56 (Fig. 10 D) and hnRNP C (Fig. 10 H), respectively, were retained within nuclei. Tungstate also induced rapid in vitro nuclear export of hsp70 (not shown). hsp90 is distributed in both the cytoplasm and nucleus (Gasc et al., 1990), indicating that it might be a shuttling protein, although this has not been experimentally verified. hsp56 is found predominantly within the nucleus (Czar et al., 1995) and its nucleocytoplasmic shuttling properties have not been assessed. The fact that molybdate and tungstate effects are exerted on hsp70, a shuttling protein, and a shuttling hnRNP protein (i.e., hnRNP A1) suggests that some component of the nuclear export machinery used by shuttling proteins may be targeted by metal oxyanions.

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