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Transient, Ligand-Dependent Arrest of the Androgen Receptor in Subnuclear Foci Alters Phosphorylation and Coactivator Interactions

Center for Cell Signaling (B.E.B., M.J.V., A.S., N.A., B.M.P.), Department of Biochemistry and Molecular Genetics (B.E.B., N.A., B.M.P.), Cell and Molecular Biology Program (B.E.B., N.A., M.J.W., B.M.P.), and Department of Microbiology (D.G., M.J.W.), Department of Health Evaluation Sciences (M.R.C.), University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Bryce M. Paschal, Ph.D, Center for Cell Signaling, Box 800577 Health Systems, University of Virginia, Charlottesville, Virginia 22908. E-mail: paschal{at}virginia.edu.

Here we report that mutations within the DNA-binding domain of AR, shown previously to inhibit nuclear export to the cytoplasm, cause an androgen-dependent defect in intranuclear trafficking of AR. Mutation of two conserved phenylalanines within the DNA recognition helix (F582, 583A) results in androgen-dependent arrest of AR in multiple subnuclear foci. A point mutation in one of the conserved phenylalanines (F582, F582Y) is known to cause androgen insensitivity syndrome (AIS). Both AIS mutants (F582, F582Y) and the export mutant (F582, 583A) displayed androgen-dependent arrest in foci, and all three mutants promoted androgen-dependent accumulation of the histone acetyl transferase CREB binding protein (CBP) in the foci. The foci correspond to a subnuclear compartment that is highly enriched for the steroid receptor coactivator glucocorticoid receptor-interacting protein (GRIP)-1. Agonist-bound wild-type AR induces the redistribution of GRIP-1 from foci to the nucleoplasm. This likely reflects a direct interaction between these proteins because mutation of a conserved residue within the major coactivator binding site on AR (K720A) inhibits AR-dependent dissociation of GRIP-1 from foci. GRIP-1 also remains foci-associated in the presence of agonist-bound F582, 583A, F582, or F582Y forms of AR. Two-dimensional phospho-peptide mapping and analysis with a phospho-specific antibody revealed that mutant forms of AR that arrest in the subnuclear foci are hypophosphorylated at Ser81, a site that normally undergoes androgen-dependent phosphorylation. Our working model is that the subnuclear foci are sites where AR undergoes ligand-dependent engagement with GRIP-1 and CBP, a recruitment step that occurs before Ser81 phosphorylation and association with promoters of target genes.

NURSA Molecule Pages Link:

Nuclear Receptors:   COUP-TFII  |  GR  |  AR

Coregulators:   CBP  |  GRIP1  |  SMRT

Ligands:   Dexamethasone  |  R1881

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