Two types of anti-progestins have distinct effects on site-specific phosphorylation of human progesterone receptor
Two types of anti-progestins have distinct effects on site-specific phosphorylation of human progesterone receptor
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DOI：
10.1074/jbc.271.2.1209
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发表时间：
1996-01-12
影响因子：
4.8
通讯作者：
Edwards, DP
中科院分区：
文献类型：
--
作者：
Beck, CA;Zhang, YX;Edwards, DP
人孕酮受体（PR）在多个丝氨酸残基上磷酸化;三个位点（Ser（102）、Ser（294）和Ser（345））可由激素激动剂诱导，而至少六个其他位点是基本磷酸化的，并且表现出对激素的响应的普遍增加。在这项研究中，我们已经使用了高效液相色谱磷酸肽图谱和手动肽测序，以探讨如何两种不同的resistin拮抗剂，RU 486和ZK 98299，影响网站特异性磷酸化PR分离T47 D乳腺癌细胞。与R5020相比，RU 486刺激每个PR分子的[P-32]磷酸的总掺入的类似增加（PR-A为2.5-2.6倍，PR-B为2.1倍），并且在位点特异性水平，RU 486刺激基础位点和诱导位点两者达到与R5020相同的程度。相比之下，ZK 98299仅产生总体磷酸化的最小增加（PR-A为1.2倍，PR-B为1.1倍），这是由于对基底位点的刺激减少，并且未能诱导三个依赖性位点中的任何一个。未检测到响应RU 486或ZK 98299的不适当磷酸化位点。在共处理研究中，ZK 98299阻断了R5020诱导的PR整体磷酸化的增加，表明这种拮抗剂刺激特定磷酸化位点的失败不是由于与完整细胞中PR的无效相互作用。这些结果表明，RU 486的生物学效应不是通过改变PR的磷酸化状态来介导的，而未能促进某些位点的磷酸化可能有助于ZK 98299的拮抗作用。此外，这些结果支持的概念，两种机制类的抗孕激素，影响PR不同的体内。
Human progesterone receptor (PR) is phosphorylated on multiple serine residues; three sites (Ser(102), Ser(294), and Ser(345)) are inducible by hormone agonist, while at least six others are basally phosphorylated and exhibit a general increase in response to hormone. In this study we have used high performance liquid chromatography phosphopeptide mapping and manual peptide sequencing to investigate how two different progestin antagonists, RU486 and ZK98299, affect site specific phosphorylation of PR isolated from T47D breast cancer cells. As compared to the progestin agonist R5020, RU486 stimulated a similar increase in overall incorporation of [P-32]phosphate per PR molecule (2.5-2.6-fold for PR-A and 2.1-fold for PR-B), and at the site-specific level, RU486 stimulated both the basal and inducible sites to the same extent as R5020. In contrast, ZK98299 produced only a minimal increase in overall phosphorylation (1.2-fold for PR-A and 1.1-fold for PR-B) which was due to a reduced stimulation of the basal sites and failure to induce any of the three hormone-dependent sites. No inappropriate phosphorylation sites were detected in response to either RU486 or ZK98299. In cotreatment studies, ZK98299 blocked the increase in overall phosphorylation of PR induced by R5020, demonstrating that the failure of this antagonist to stimulate specific phosphorylation sites is not due to an inefficient interaction with PR in the intact cell. These results indicate that the biological effects of RU486 are not mediated by an alteration in the phosphorylation state of PR, whereas failure to promote phosphorylation of certain sites may contribute to the antagonist action of ZK98299. Additionally these results support the concept of two mechanistic classes of anti-progestins that affect PR differently in vivo.