Targeting KCTD13 and β1Pix Enhance Androgen Receptor Ubiquitination and Degradation
Abhishek Seth, MD1, Carolina Jorgez, PhD2, Ahmed Chahdi, PhD1.
1Nemours Children's Health Orlando, orlando, FL, USA, 2Baylor College of Medicine, Houston, TX, USA.
BACKGROUND:Congenital anomalies of the genitourinary (GU) tract are a common occurrence that arise from disturbances during embryonic development. These GU anomalies include hypospadias and cryptorchidism. Hypospadias is the improper positioning of the urethral opening on the ventral side of the penis. Cryptorchidism, or undescended testis (UDT), is the most common GU birth defect. We previously demonstrated the importance of KCTD13 in the GU tract development and in androgen receptor (AR) signaling. KCTD13 is a substrate-specific adapter of the CUL3-based ubiquitin ligase complex. The underlying molecular mechanisms of these congenital GU defects are not well understood. The objective of the present study is to determine the role of KCTD13 and β1Pix (p21-activated kinase (PAK)-interacting exchange factor β) in AR ubiquitination and degradation. β1Pix is a Rac/Cdc42 guanine nucleotide exchange factor (GEF) responsible for the recruitment and activation of Rho GTPases at the cell membrane.
METHODS:Cell culture, immunoblotting, co-immunoprecipitation, immunohistochemistry, cell fractionation and luciferase assays were used to study the role of KCTD13 in AR ubiquitination/degradation.
RESULTS:Ablation of the Kctd13 gene reduced the protein levels of AR and βPix, and increased AR ubiquitination in mouse testicular tissues. We show here that the Rac/Cdc42 guanosine triphosphatase guanine nucleotide exchange factor, β1Pix (Pak-interacting exchange factor) enhanced AR protein levels induced by AR stimulation in HEK293 and HeLa cells. βPix and Kctd13 increase AR stability and decrease AR ubiquitination. AR stimulation increased β1Pix binding to AR which is accompanied by decreased AR ubiquitination. Ectopic expression of β1Pix and Kctd13 decreased AR ubiquitination. Furthermore, we demonstrate the formation of a multimolecular complex comprised of AR/β1Pix/KCTD13/STUB1. β1Pix and/or KCTD13 binding dissociates STUB1 from AR and thus prevents STUB1 from catalyzing receptor ubiquitination., suggesting that β1Pix/KCTD13 signaling is an important modulator of ligand-dependent AR transcriptional activity. We propose that β1Pix/KCTD13 could serve as a promising therapeutic target to modulate AR availability and/or AR signaling.
CONCLUSIONS:Collectively, our data reveal a novel signaling complex comprised of AR-β1Pix-KCTD13-STUB1 through which β1Pix and KCTD13 are critical in maintaining AR stability and signaling by preventing AR ubiquitination and proteasomal degradation
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