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Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
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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