Joel P B, Traish A M, Lannigan D A
Department of Biology, University of Vermont, Burlington 05405, USA.
Mol Endocrinol. 1995 Aug;9(8):1041-52. doi: 10.1210/mend.9.8.7476978.
Serine 118 is definitively identified as a major site of phosphorylation in the human estrogen receptor expressed in COS-1 cells treated with estradiol or phorbol ester. At least 30% of the estrogen receptor appears to be phosphorylated on serine 118 after treatment with estradiol or phorbol ester. Human estrogen receptor was expressed in COS-1 cells and labeled in vivo with [32P]orthophosphate in the presence of estradiol or phorbol ester. Immunopurified receptor was digested with cyanogen bromide. The most heavily labeled peptide (7 kilodaltons) was identified as amino acids 110-174 by microsequencing. Manual Edman degradation released a major portion of the 32P-label in the peptide at serine 118. A mutant with serine 118 replaced by alanine (S118A) had 80% less 32P-label in the 7 kilodalton peptide. Estrogen receptor labeled in vivo with [32P]-orthophosphate in the presence of estradiol or phorbol ester migrates electrophoretically as a doublet. The major difference between the bands is phosphorylation of serine 118 in the upshifted band. The mutant S118A does not show an upshifted band. Labeling of the estrogen receptor with [35S]methionine indicates that > or = 30% of the receptor is upshifted and suggests that > or = 30% of the receptor is phosphorylated on serine 118.
丝氨酸118被明确鉴定为在用雌二醇或佛波酯处理的COS-1细胞中表达的人雌激素受体的主要磷酸化位点。在用雌二醇或佛波酯处理后，至少30%的雌激素受体似乎在丝氨酸118上发生了磷酸化。人雌激素受体在COS-1细胞中表达，并在雌二醇或佛波酯存在的情况下用[32P]正磷酸盐进行体内标记。免疫纯化的受体用溴化氰消化。通过微测序将标记最强烈的肽（7千道尔顿）鉴定为氨基酸110-174。手动埃德曼降解在丝氨酸118处释放了该肽中大部分的32P标记。丝氨酸118被丙氨酸取代的突变体（S118A）在7千道尔顿的肽中的32P标记减少了80%。在雌二醇或佛波酯存在的情况下用[32P]正磷酸盐进行体内标记的雌激素受体在电泳时迁移为双峰。两条带之间的主要差异是上移带中丝氨酸118的磷酸化。突变体S118A没有显示出上移带。用[35S]甲硫氨酸对雌激素受体进行标记表明，≥30%的受体发生了上移，这表明≥30%的受体在丝氨酸118上发生了磷酸化。
