Ligand Binding to the Androgen Receptor Induces Conformational Changes That Regulate Phosphatase Interactions

Abstract

We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen-and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation.The nuclear receptor superfamily of transcription factors directs the expression of genes whose products regulate diverse biological pathways. The domain organization of nuclear receptors is conserved and includes an N-terminal activation function 1 (AF-1) region, a central DNA binding domain (DBD), and a C-terminal ligand-binding domain (LBD). Ligand binding to the LBD initiates a series of changes in nuclear receptor structure, chaperone composition, localization, transactivation potential, and protein half-life (t 1/2 ) (10,28,36,44). Understanding how ligand binding elicits these changes is fundamental to understanding nuclear receptor regulation and activity. Defining how ligands control nuclear receptor activity should also provide insight into certain disease mechanisms and aid in the design of drugs such as selective androgen receptor modulators and selective estrogen receptor modulators (4, 27).In addition to ligand binding, nuclear receptors can be regulated by signal transduction pathways. Kinases including those controlled by growth factor-dependent pathways act directly or indirectly on a variety of nuclear receptors (37). Kinases reported to regulate androgen receptor (AR)-dependent transcription include the mitogen-activated protein kinases (p42/ 44, p38, and Jun N-terminal protein kinase), protein kinase A, and protein kinase C (8). The mitogen-activated protein kinases p38 and Jun N-terminal protein kinase also regulate the nucleocytoplasmic distribution of AR (15). Determining exactly how kinases regulate nuclear receptor transcription activity has been challenging because of cross talk bet...

Activation of the DNA-dependent Protein Kinase Stimulates Nuclear Export of the Androgen Receptor in Vitro

“…This suggests that direct phosphorylation of AR could be one of the ratelimiting steps for AR export in vitro. AR is phosphorylated on multiple serines (54,55) and on at least one tyrosine (56). Phosphorylation of Ser-650 in the hinge region positively regulates AR internuclear migration in a heterokaryon assay (27).…”

FKBP51 Promotes Assembly of the Hsp90 Chaperone Complex and Regulates Androgen Receptor Signaling in Prostate Cancer Cells

“…AR transcription was measured using the dual-luciferase reporter system and plotted as firefly/Renilla as described previously (35). The firefly luciferase reporter genes used to measure AR activity were based on the promoters for prostate-specific antigen (PSA), probasin, mouse mammary tumor virus (MMTV), and synthetic androgen response elements (AREs).…”

“…Changes in the number of AR molecules that undergo androgen binding in response to FKBP51 expression were also assessed by using the AR-PP2A interaction, which is strictly dependent on the agonist conformation of AR (35). PC-3 cells were cotransfected with expression plasmids including pcDNA3-Flag-AR and simian virus 40 (SV40) small t antigen with either PCI-FKBP51 or a control plasmid.…”

“…The following antibodies were used for immunoblotting at a dilution of 1:1,000 unless noted otherwise: Hsp90 (H90-10) (2), Hsp70 (BB70) (32), p23 (JJ3) (10), FKBP51 (Hi51B), FKBP52 (Hi52A) (18), PP2Ac (ID6; Upstate), AR (AR21, 1:5,000) (35), and PSA (AR014-5RE, 1:100; Biogenix). FKBP51 (clone 18, 1:20; BD Transduction) was used for immunocytochemistry.…”

Final steps in the feedback regulation of human glucocorticoid receptor gene and role of nuclear protein phosphatase 2A

“…This is further reflected by the fact that a GR AF-1 specific phosphatase (GeneBank Accession # AF74498), which varies considerably from PPP2R3C, does not de-phosphorylate CREB-P. These results have potential to be useful in the designing of diagnostic regimen for patients who become resistant to glucocorticoid therapy during the course of their treatment [26]. Resistance, which is the result of a loss in GR function, due to down-regulation of GR expression leads to decreased sensitivity to Dex, a serious complication in the treatment of arthritis and severe asthma.…”