Abstract
The androgen receptor (AR) is a ligand-activated nuclear receptor that plays a critical role in normal prostate physiology, as well as in the development and progression of prostate cancer. In addition to the classical paradigm in which AR exerts its biological effects in the nucleus by orchestrating the expression of the androgen-regulated transcriptome, there is considerable evidence supporting a rapid, nongenomic activity mediated by membrane-associated AR. Although the genomic action of AR has been studied in depth, the molecular events governing AR transport to the plasma membrane and the downstream AR signaling cascades remain poorly understood. In this study, we report that AR membrane transport is microtubule-dependent. Disruption of the function of kinesin 5B (KIF5B), but not of kinesin C3 (KIFC3), interfered with AR membrane association and signaling. Co-immunoprecipitation and pulldown assays revealed that AR physically interacts with KIF5B and that androgen enhances this interaction. Furthermore, we show that heat shock protein 27 (HSP27) is activated by membrane-associated AR and that HSP27 plays an important role in mediating AR-mediated membrane-to-nuclear signal transduction. Together, these results indicate that AR membrane translocation is mediated by the microtubule cytoskeleton and the motor protein KIF5B. By activating HSP27, membrane-associated AR potentiates the transcriptional activity of nuclear AR. We conclude that disruption of AR membrane translocation may represent a potential strategy for targeting AR signaling therapeutically in prostate cancer.
Highlights
The androgen receptor (AR) is a ligand-activated nuclear receptor that plays a critical role in normal prostate physiology, as well as in the development and progression of prostate cancer
Similar observations were made in COS-7 cells transfected with AR (Fig. S1), suggesting AR membrane translocation is not unique to LNCaP cells
As a member of the nuclear receptor superfamily, AR is best known for its activity in the nucleus as a ligand-dependent transcription factor, orchestrating the expression of androgen-regulated transcriptome which is critical for prostate development, homeostasis, and carcinogenesis [1, 15, 28]
Summary
We first employed an immunofluorescence (IF) assay to visualize androgen-induced AR membrane localization in LNCaP cells. At 20 min, as the majority of cytoplasmic AR had translocated to the nucleus, the membrane pool became more distinguishable. This fraction of AR appeared to peak at around 20 min after androgen stimulation, as the intensity declined after longer incubations. Consistent with a previous report showing palmitoylation is critical for AR membrane translocation [10], treatment with 2-bromopalmitate (2-BP), a palmitoylation inhibitor, blocked the androgen-induced increase of AR in the TI fraction (Fig. S3). AR was present at a very low level in Gi␣3-positive fractions under the androgen-depleted condition, and treatment with androgen significantly increased AR distribution in these fractions (Fig. 1C).
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