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Calmodulin-Androgen Receptor (AR) Interaction: Calcium-Dependent, Calpain-Mediated Breakdown of AR in LNCaP Prostate Cancer Cells

¹ Vattikuti Urology Institute and ² Department of Dermatology, Henry Ford Hospital; ³ Karmanos Cancer Institute and Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan

Requests for reprints: G. Prem-Veer Reddy, Vattikuti Urology Institute, Henry Ford Health System, One Ford Place, 2D, Detroit, MI 48202. Phone: 313-874-5991; Fax: 313-874-4324; E-mail: preddy1{at}hfhs.org.

Chemotherapy of prostate cancer targets androgen receptor (AR) by androgen ablation or antiandrogens, but unfortunately, it is not curative. Our attack on prostate cancer envisions the proteolytic elimination of AR, which requires a fuller understanding of AR turnover. We showed previously that calmodulin (CaM) binds to AR with important consequences for AR stability and function. To examine the involvement of Ca²⁺/CaM in the proteolytic breakdown of AR, we analyzed LNCaP cell extracts that bind to a CaM affinity column for the presence of low molecular weight forms of AR (intact AR size, 114 kDa). Using an antibody directed against the NH₂-terminal domain (ATD) of AR on Western blots, we identified 76-kDa, 50-kDa, and 34/31-kDa polypeptides in eluates of CaM affinity columns, suggesting the presence of CaM-binding sites within the 31/34-kDa ATD of AR. Under cell-free conditions in the presence of phenylmethylsulfonyl fluoride, AR underwent Ca²⁺-dependent degradation. AR degradation was inhibited by N-acetyl-leu-leu-norleu, an inhibitor of thiol proteases, suggesting the involvement of calpain. In intact cells, AR breakdown was accelerated by raising intracellular Ca²⁺ using calcimycin, and increased AR breakdown was reversed with the cell-permeable Ca²⁺ chelator bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester. In CaM affinity chromatography studies, the Ca²⁺-dependent protease calpain was bound to and eluted from the CaM-agarose column along with AR. Caspase-3, which plays a role in AR turnover under stress conditions, did not bind to the CaM column and was present in the proenzyme form. Similarly, AR immunoprecipitates prepared from whole-cell extracts of exponentially growing LNCaP cells contained both calpain and calpastatin. Nuclear levels of calpain and calpastatin (its endogenous inhibitor) changed in a reciprocal fashion as synchronized LNCaP cells progressed from G₁ to S phase. These reciprocal changes correlated with changes in AR level, which increased in late G₁ phase and decreased as S phase progressed. Taken together, these observations suggest potential involvement of AR-bound CaM in calcium-controlled, calpain-mediated breakdown of AR in prostate cancer cells. (Cancer Res 2006; 66(24): 11754-62)

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