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The role of selective degradation of AR-V7 through KCTD13-CUL3 complex in masculinization
AHMED CHAHDI, PhD1, Carolina Jorgez, PhD2, Abhishek Seth, MD1.
1Nemours Children's Hospital, Orlando, FL, USA, 2Scott Department of Urology, Baylor College of Medicine, Houston, TX, USA.

Background: The 16p11.2 deletion and duplication syndromes are associated with a wide spectrum of abnormal developmental phenotypes. Copy number variations (CNV) are an important risk factor for congenital genitourinary defects. One of the most common CNVs involved in congenital genitourinary anomalies is located at the 16p11.2 locus. We identified KCTD13 as an important gene in this region that may be associated with male lower genitourinary tract defects. KCTD13 is a substrate-specific adapter of the E3 ubiquitin ligase CULLIN-3 (CUL-3). Androgen signaling is mediated by the nuclear androgen receptor (AR), which plays fundamental roles in masculinization during development. Androgen signaling-induced mesenchymal cell proliferation has been observed in masculinization. Such action is essential for regulating urogenital tissue development, including external genitalia development. AR splice variant 7 (AR-V7) is a ligand-independent and constitutively activating variant of AR and is considered as the key driver to initiate castration-resistant prostate cancer. We hypothesize that, like the mouse model, alterations in the KCTD13-CUL3 complex may affect the masculinization axis in boys through inefficient ubiquitination of the masculinization pathways. Methods: Equal amounts (500 g) of testis homogenates from WT and Kctd13-/- mice were immunoprecipitated using 4 g of anti-AR or anti-AR-V7 antibodies for 2 h followed by incubation with 30 L of protein A/G agarose with constant rotation for an additional hour. The immunoprecipitants were washed 3 times and were eluted with 30 L of SDS-PAGE sample buffer by boiling for 5 min. The eluted proteins were electrophoresed on 8% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against ubiquitin, CUL-3, Hsp70, Hsp90, AR and AR-V7. To determine the effect of Kctd13 gene deletion on the levels of target proteins, 20 g of each homogenate from WT and Kctd13-/- mice were loaded into a 4-12% SDS-PAGE and the proteins were revealed using antibodies against CUL-3, Hsp70, Hsp90, AR, AR-V7 and β actin. Results: We demonstrate that the deletion of Kctd13 gene in mice increased the expression of Hsp90 and CUL-3 while Hsp70 and AR levels were decreased. AR splice variant 7 (AR-V7), a ligand-independent and constitutively activating variant of AR, is considered as the key driver to initiate castration-resistant prostate cancer. Our data show that in WT mice, CUL-3 binds specifically to AR-V7, but not AR, leading to AR-V7 ubiquitination and degradation. More importantly, CUL-3 binding to and ubiquitination of AR-V7 is suppressed in Kctd13-/- mice. Furthermore, KCTD13-deficient mice exhibit testicular and penile abnormalities together with significantly reduced levels of nuclear AR. In Vitro, KCTD13 knockdown results in significantly decreased levels of nuclear AR, suggesting that loss of KCTD13 affects AR sub-cellular localization. Conclusion: Together, our results show that Kctd13 deletion 1) increased binding of CUL-3 to AR-V7 resulting in AR-V7 proteasomal ubiquitination/degradation and 2) decreased Hsp70 levels inhibits AR transcriptional activity. Our data reveal a novel mechanism explaining the critical role, through ubiquitination/degradation alteration, of Kctd13 in regulating the virilization mediated by the AR signaling axis.


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