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 Ca(2+)/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, approximately 114 kDa). Using an antibody directed against the NH(2)-terminal domain (ATD) of AR on Western blots, we identified approximately 76-kDa, approximately 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 Ca(2+)-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 Ca(2+) using
calcimycin, and increased AR breakdown was reversed with the cell-permeable Ca(2+)
chelator bis-(O-aminophenoxy)-
ethane-N,N,N',N'-tetraacetic
acid tetra-(acetoxymethyl)-
ester. In CaM affinity chromatography studies, the Ca(2+)-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 G(1) to S phase. These reciprocal changes correlated with changes in AR level, which increased in late G(1) 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.