Transient, ligand-dependent arrest of the androgen receptor in subnuclear foci alters phosphorylation and coactivator interactions.
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 (DeltaF582, F582Y) is known to cause androgen insensitivity syndrome (AIS). Both AIS mutants (DeltaF582, 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, DeltaF582, 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.
Black
Ben E
BE
Center for Cell Signaling, Department of Biochemistry and Molecular Genetics, Box 800577 Health Systems, University of Virginia, Charlottesville, VA 22908, USA.
Vitto
Michael J
MJ
Gioeli
Daniel
D
Spencer
Adam
A
Afshar
Nima
N
Conaway
Mark R
MR
Weber
Michael J
MJ
Paschal
Bryce M
BM
D013734:Androgen-Insensitivity Syndrome / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006801:Humans; D008024:Ligands; D008297:Male; D009154:Mutation; D051958:Nuclear Receptor Coactivator 2; D010766:Phosphorylation; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*
D008024:Ligands; C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D011944:Receptors, Androgen; D014157:Transcription Factors