Calmodulin-androgen receptor (AR) interaction: Calcium-dependent, calpain-mediated breakdown of AR in LNCaP prostate cancer cells

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 Aft (intact All size, similar to 114 kDa). Using an antibody directed against the NH2-terminal domain (ATD) of AR on Western blots, we identified similar to 76-kDa, similar to 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 All. 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 wholecell 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 G, to S phase. These reciprocal changes correlated with changes in AR level, which increased in late G, phase and decreased as S phase progressed. Taken together, these observations suggest potential involvement of AR-bound CaM in calciumcontrolled, calpain-mediated breakdown of AR in prostate cancer cells.