TY - JOUR
T1 - Androgens Negatively Regulate Forkhead Transcription Factor FKHR (FOXO1) Through A Proteolytic Mechanism in Prostate Cancer Cells
AU - Huang, Haojie
AU - Muddiman, David C.
AU - Tindall, Donald J.
PY - 2004/4/2
Y1 - 2004/4/2
N2 - The ability of androgens to inhibit apoptosis in both normal and malignant prostatic cells has been well documented. However, the underlying mechanisms are understood poorly. Here we demonstrated that forkhead transcription factor FKHR (FOXO1)-induced death of LNCaP cells was blocked by a synthetic androgen R1881. Androgen treatment also resulted in a reduction in transcriptional activity of FKHR in these cells. Moreover, treatment of LNCaP cells with R1881 led to a decrease in the intact FKHR protein (70 kDa) and an increase in a faster migrating protein band (60 kDa). Androgen-enhanced appearance of the 60-kDa protein was diminished specifically by lysosomal acidic cysteine protease inhibitors. Mass spectrometry analyses of the purified FLAG-tagged 70- and 60-kDa proteins demonstrated that the 60-kDa species is a FKHR protein product that lacks about 120 amino acid residues of the C-terminal end. Mutagenesis of the basic amino acid Arg537 in the protease cleavage region, as suggested by mass spectrometry, abrogated both the androgen-induced accumulation of the 60-kDa product and decrease in cell death induced by FKHR, suggesting that the residue Arg537 is a potential protease cleavage site. Finally, ectopic expression of the first 537 amino acids of FKHR produced an inhibitory effect on transcriptional activity of the intact protein. Together, these results suggest that androgens induce increased activity of an acidic cysteine protease, which in turn cleaves FKHR. This provides a mechanism by which androgens protect prostate cancer cells from the killing effect of FKHR.
AB - The ability of androgens to inhibit apoptosis in both normal and malignant prostatic cells has been well documented. However, the underlying mechanisms are understood poorly. Here we demonstrated that forkhead transcription factor FKHR (FOXO1)-induced death of LNCaP cells was blocked by a synthetic androgen R1881. Androgen treatment also resulted in a reduction in transcriptional activity of FKHR in these cells. Moreover, treatment of LNCaP cells with R1881 led to a decrease in the intact FKHR protein (70 kDa) and an increase in a faster migrating protein band (60 kDa). Androgen-enhanced appearance of the 60-kDa protein was diminished specifically by lysosomal acidic cysteine protease inhibitors. Mass spectrometry analyses of the purified FLAG-tagged 70- and 60-kDa proteins demonstrated that the 60-kDa species is a FKHR protein product that lacks about 120 amino acid residues of the C-terminal end. Mutagenesis of the basic amino acid Arg537 in the protease cleavage region, as suggested by mass spectrometry, abrogated both the androgen-induced accumulation of the 60-kDa product and decrease in cell death induced by FKHR, suggesting that the residue Arg537 is a potential protease cleavage site. Finally, ectopic expression of the first 537 amino acids of FKHR produced an inhibitory effect on transcriptional activity of the intact protein. Together, these results suggest that androgens induce increased activity of an acidic cysteine protease, which in turn cleaves FKHR. This provides a mechanism by which androgens protect prostate cancer cells from the killing effect of FKHR.
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U2 - 10.1074/jbc.M314143200
DO - 10.1074/jbc.M314143200
M3 - Article
C2 - 14726521
AN - SCOPUS:1842690558
SN - 0021-9258
VL - 279
SP - 13866
EP - 13877
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -