Rajbhandari Prashant, Ozers Mary Szatkowski, Solodin Natalia M, Warren Christopher L, Alarid Elaine T
From the McArdle Laboratories for Cancer Research, Department of Oncology and University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, Wisconsin 53705 and.
Proteovista LLC, Madison, Wisconsin 53719.
J Biol Chem. 2015 May 29;290(22):13749-62. doi: 10.1074/jbc.M114.621698. Epub 2015 Apr 12.
The transcriptional activity of estrogen receptor α (ERα), the key driver of breast cancer proliferation, is enhanced by multiple cellular interactions, including phosphorylation-dependent interaction with Pin1, a proline isomerase, which mediates cis-trans isomerization of the N-terminal Ser(P)(118)-Pro(119) in the intrinsically disordered AF1 (activation function 1) domain of ERα. Because both ERα and Pin1 have multiple cellular partners, it is unclear how Pin1 assists in the regulation of ERα transactivation mechanisms and whether the functional effects of Pin1 on ERα signaling are direct or indirect. Here, we tested the specific action of Pin1 on an essential step in ERα transactivation, binding to specific DNA sites. DNA binding analysis demonstrates that stable overexpression of Pin1 increases endogenous ERα DNA binding activity when activated by estrogen but not by tamoxifen or EGF. Increased DNA binding affinity is a direct effect of Pin1 on ERα because it is observed in solution-based assays with purified components. Further, our data indicate that isomerization is required for Pin1-modulation of ERα-DNA interactions. In an unbiased in vitro DNA binding microarray with hundreds of thousands of permutations of ERα-binding elements, Pin1 selectively enhances the binding affinity of ERα to consensus DNA elements. These studies reveal that Pin1 isomerization of phosphorylated ERα can directly regulate the function of the adjacent DNA binding domain, and this interaction is further modulated by ligand binding in the ligand-binding domain, providing evidence for Pin1-dependent allosteric regulation of ERα function.
雌激素受体α（ERα）是乳腺癌增殖的关键驱动因子，其转录活性通过多种细胞相互作用得以增强，包括与脯氨酸异构酶Pin1的磷酸化依赖性相互作用，Pin1介导ERα内在无序的AF1（激活功能1）结构域中N端Ser(P)(118)-Pro(119)的顺反异构化。由于ERα和Pin1都有多个细胞伴侣，目前尚不清楚Pin1如何协助调节ERα的反式激活机制，以及Pin1对ERα信号传导的功能影响是直接的还是间接的。在这里，我们测试了Pin1对ERα反式激活中一个关键步骤（即与特定DNA位点结合）的特定作用。DNA结合分析表明，Pin1的稳定过表达在雌激素激活时增加内源性ERα的DNA结合活性，但在他莫昔芬或表皮生长因子（EGF）激活时则不然。DNA结合亲和力的增加是Pin1对ERα的直接作用，因为在基于纯化成分的溶液分析中观察到了这一点。此外，我们的数据表明异构化是Pin1调节ERα-DNA相互作用所必需的。在一个具有数十万个ERα结合元件排列的无偏体外DNA结合微阵列中，Pin1选择性地增强了ERα与共有DNA元件的结合亲和力。这些研究表明，磷酸化的ERα的Pin1异构化可以直接调节相邻DNA结合结构域的功能，并且这种相互作用在配体结合结构域中被配体结合进一步调节，为Pin1依赖性的ERα功能变构调节提供了证据。
