PHOSPHORYLATION OF TYROSINE-537 ON THE HUMAN ESTROGEN-RECEPTOR IS REQUIRED FOR BINDING TO AN ESTROGEN RESPONSE ELEMENT

被引:0
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作者
ARNOLD, SF
VOROJEIKINA, DP
NOTIDES, AC
机构
[1] UNIV ROCHESTER,SCH MED & DENT,DEPT ENVIRONM MED,ROCHESTER,NY 14642
[2] UNIV ROCHESTER,SCH MED & DENT,DEPT BIOPHYS,ROCHESTER,NY 14642
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中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We report here that the phosphorylation of tyrosine 537 on the human estrogen receptor (hER) controls the receptor's dimerization and DNA binding ability. The DNA-binding form of both the hER from human MCF-7 mammary carcinoma cells and the hER overexpressed in Sf9 insect cells was isolated using estrogen response element (ERE) affinity chromatography. Western blot analyses demonstrated that the DNA-binding form of the hER from MCF-7 or Sf9 cells was (i) phosphorylated at tyrosine 537, (ii) localized in the nucleus of estradiol-treated MCF-7 cells with an apparent molecular mass of 67 kDa, and (iii) hyperphosphorylated at serine residue(s). The non-DNA-binding form of the hER was (i) devoid of phosphorylation at tyrosine 537, (ii) cytosolic with an apparent molecular mass of 66 kDa, and (iii) hypophosphorylated at serine residue(s). The dephosphorylation of the purified hER at phosphotyrosine 537 with a tyrosine phosphatase eliminated binding to an ERE in a gel mobility shift assay. The binding of the tyrosine-dephosphorylated hER to an ERE was restored by the rephosphorylation of tyrosine 537 with Src family tyrosine kinases, p60(c-src) or p56(lck), Mutation of tyrosine 537 to phenylalanine confirmed that the phosphorylation of tyrosine 537 is necessary for the hER to bind an ERE. An anti-hER antibody restored the binding of the tyrosine-dephosphorylated hER to an ERE, indicating that the bivalent anti-hER antibody brought together the two inactive hER monomers. A far-Western blot confirmed that phosphotyrosine 537 is required for hER homodimerization. These experiments establish that dimerization of the hER and DNA binding are regulated by phosphorylation at tyrosine 537. This is the first demonstration of the regulation of dimerization of a steroid hormone receptor by phosphorylation. These results are significant since p60(c-src) is overexpressed in estrogen-dependent breast cancers and may act to enhance the activity of the hER.
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页码:30205 / 30212
页数:8
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