PHOSPHORYLATION OF BREAST CANCER PROGESTERONE RECEPTORS

类固醇受体属于蛋白质超家族，除了 经典的类固醇激素，也结合维生素D3，维甲酸和 甲状腺激素。当被激素占据时，受体与它们的同源物结合。 DNA反应元件，并作为增强剂调节基因转录。 到目前为止，所有研究的类固醇受体都是磷蛋白，但功能 对磷酸化的影响尚不清楚。我们已经提纯了人孕酮 受体(PR)，并制作了针对它们的克隆抗体。有两个 人PR：120 kDa的B受体和94 kDa的A受体。通过 免疫亲和纯化，凝胶电泳，免疫印迹， 放射自显影，化学裂解，磷酸胰解肽图谱，以及 反相高压液相色谱，我们已经证明在 培养的乳腺癌细胞，当PR在多个位置被磷酸化时 没有被荷尔蒙占据的。这代表基础磷酸化。一秒钟， 激素依赖性的磷酸化在孕激素的5分钟内发生 治疗细胞，并增加比活性的细胞 [32P]正磷酸盐标记5-10倍。PR的磷酸化是在丝氨酸上 残基主要位于受体的氨基末端的一半。 在此应用程序中，我们计划精确绘制所涉及的丝氨酸残基 在PR磷酸化和确定磷酸化功能中：AIM 一是对酶促磷肽片段的氨基酸进行了测序 从高效液相色谱中洗脱出来，并将这些肽放在全长人体的上下文中 PR蛋白。目的2是为了获得A-和B-的一系列cDNA突变体 感受器。首先，删除氨基中的三个磷酸化簇- 通过突变编码B-和A-的全长hpr cDNA的末端 受体，或删除三个主要功能域并测试 基础和激素依赖性磷酸化的突变受体。第二, 利用寡核苷酸定向的定点突变保守治疗 将关键的丝氨酸残基突变为丙氨酸，并测试突变的受体 磷酸化与反式激活孕激素反应报告基因的能力 基因。第三，测试简单启动子和复杂启动子上的突变受体 评价磷酸化的功能。迭代周期数 突变的可能性应该集中在突变被消灭或 改变受体功能。在目标3中，DNA结合受体突变体将 构建以评估PR磷酸化的DNA结合要求。 这些研究将描绘PR磷酸化的功能，定义 A和B受体在反式激活中的特定作用，并作为一种 其他类固醇受体的磷酸化作用模型和 转录因子。

Steroid receptors belong to a superfamily of proteins that, in addition to the classic steroid hormones, also bind vitamin D3, retinoic acid and thyroid hormone. When occupied by hormone, receptors bind to their cognate DNA response elements and act as enhancers to regulate gene transcription. All steroid receptors studied to date are phosphoproteins but the function of phosphorylation is unknown. We have purified human progesterone receptors (PR) and have made monoclonal antibodies to them. There are two human PRs: B-receptors of 120 kDa and A-receptors of 94 kDa. By immunoaffinity purification, gel electrophoresis, immunoblotting, autoradiography, chemical cleavage, phosphotryptic peptide mapping, and reverse-phase high pressure liquid chromatography, we have shown that in cultured breast cancer cells, PRs are phosphorylated at multiple sites when unoccupied by hormone. This represents basal phosphorylation. A second, hormone-dependent phosphorylation occurs within 5 min of progestin treatment of cells, and increases the specific activity of [32P]orthophosphate labeling 5-10 fold. PR phosphorylation is on serine residues located predominately in the amino-terminal half of the receptors. In this application we plan to precisely map the serine residues involved in PR phosphorylation and determine the function of phosphorylation: Aim 1 is to sequence the amino acids of enzymatic phosphopeptide fragments eluted from HPLC and place the peptides in context on the full-length human PR protein. Aim 2 is to generate a series of cDNA mutants of the A- and B- receptors. First, to delete three phosphorylated clusters in the amino- terminus by mutagenesis of full-length hPR cDNAs encoding B- and A- receptors, or to delete the three major functional domains and test the mutant receptors for basal and hormone-dependent phosphorylation. Second, to use oligonucleotide-directed site specific mutagenesis to conservatively mutate key serine residues to alanine, and test the mutant receptors for phosphorylation and ability to trans-activate progestin responsive reporter genes. Third, to test the mutant receptors on simple and complex promoters to evaluate the function of phosphorylation. A number of iterative cycles of mutagenesis should converge on the residues whose mutation abrogates or modifies receptor function. In Aim 3, DNA binding receptor mutants will be constructed to evaluate the DNA binding requirement for PR phosphorylation. These studies will delineate the function of PR phosphorylation, define the specific roles of A- and B-receptors in transactivation, and serve as a model for the role of phosphorylation of other steroid receptors and transcription factors.