Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent and DNA-dependent stages: analysis with zinc finger mutants and the progesterone antagonist ZK98299.
Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent and DNA-dependent stages: analysis with zinc finger mutants and the progesterone antagonist ZK98299.
复制标题
激素诱导的孕酮受体磷酸化由连续的 DNA 独立和 DNA 依赖阶段组成：用锌指突变体和孕酮拮抗剂 ZK98299 进行分析。
DOI：
10.1073/pnas.89.7.3050
复制
发表时间：
1992
影响因子：
11.1
通讯作者：
Horwitz,KB
中科院分区：
文献类型：
--
作者：
Takimoto,GS;Tasset,DM;Eppert,AC;Horwitz,KB
人孕酮受体（hPR）在多个丝氨酸残基处磷酸化，首先在基础步骤中，然后在酶诱导步骤中。为了确定hPR诱导的磷酸化是在hPR与DNA相互作用之前还是之后，使用了两种策略。(i)DNA结合被阻止或改变与位点特异性突变体的A型的人孕激素受体;（ii）DNA结合的野生型人孕激素受体的形式A和B被阻止与孕酮拮抗剂ZK 98299。构建了两种hPRA突变体：DBDCys，其在第一锌指中缺乏关键的半胱氨酸残基，和DBDsp，其在三个区别性氨基酸处突变以将其DNA结合特异性从孕酮反应元件改变为雌激素反应元件。受体在PR阴性细胞中瞬时表达，并位于核内。DBDCys在体外不结合DNA，DBDsp仅结合雌激素反应元件。瞬时表达的hPRA和DBDsp表现出电泳迁移率的上移特征的蛋白酶诱导的磷酸化，它是不存在的DBDCys。与hPRA和DBDsp相比，激素诱导的[32 P]正磷酸盐掺入瞬时表达的DBDCys减少了60%，但没有消除。ZK 98299结合hPR，但阻止它们与DNA的相互作用。与R5020相比，拮抗剂使T47 D乳腺癌细胞中hPRB和hPRA的磷酸化降低了60%，并完全阻止了迁移率的改变。我们的结论是，酶诱导的磷酸化的hPR包括DNA独立和DNA依赖的阶段，只有DNA依赖的网站有助于迁移率的转变。
Human progesterone receptors (hPRs) are phosphorylated at multiple serine residues, first in a basal step and then in a hormone-induced step. To determine whether hormone-induced phosphorylation precedes or follows the interaction of hPRs with DNA two strategies were used. (i) DNA binding was prevented or altered with site-specific mutants of the A form of hPR; (ii) DNA binding of wild-type hPR forms A and B was prevented with the progesterone antagonist ZK98299. Two hPRA mutants were constructed: DBDCys, which lacks a critical cysteine residue in the first zinc finger, and DBDsp, which is mutated at three discriminatory amino acids to change its DNA binding specificity from a progesterone response element to an estrogen response element. Receptors were transiently expressed in PR-negative cells and were intranuclear. DBDCys did not bind DNA in vitro and DBDsp bound only the estrogen response element. Transiently expressed hPRA and DBDsp showed the upward shift in electrophoretic mobility characteristic of hormone-induced phosphorylation; it was absent with DBDCys. Hormone-induced [32P] orthophosphate incorporation into transiently expressed DBDCys was reduced 60% compared to hPRA and DBDsp but was not eliminated. ZK98299 binds hPRs but prevents their interaction with DNA. Compared to R5020, the antagonist reduced phosphorylation of hPRB and hPRA in T47D breast cancer cells by 60% and totally prevented the mobility shift. We conclude that the hormone-induced phosphorylation of hPR includes DNA-independent and DNA-dependent stages and that only DNA-dependent sites contribute to the mobility shift.