Localization and hormonal stimulation of phosphorylation sites in the LNCaP-cell androgen receptor

Abstract

Phosphorylation of the androgen receptor in human prostate tumour cells (LNCaP) is increased by addition of androgens to intact cells. Double-label studies, using [35S]methionine incorporation into receptor protein, and [32P]P1 to label metabolically receptor phosphorylation sites, have enabled us to determine the phosphate content, relative to receptor protein, of both non-transformed and transformed androgen receptors generated in intact LNCaP cells. No net change in the phosphorylation of the intact 110kDa steroid-binding component of the androgen-receptor complex was found upon transformation to the tight nuclear binding form in the intact cell. Partial proteolysis of androgen receptor protein metabolically labelled with [32P]P(i) and photolabelled with [3H]R1881 (methyltrienolone) revealed that phosphorylation occurs mainly in the N-terminal trans-activation domain, whereas no phosphorylation was detected in the steroid- and DNA-binding domains. The location of most (> 90%) of the hormonally regulated phosphorylation sites in the N-terminal trans-activation domain suggests a role of phosphorylation of the androgen receptor in transcription regulation.