Multimeric Signaling Complexes in PRLr Transduction

描述（由申请人提供）：催乳素（PRL）是一种用于正常和恶性乳腺组织的激素，其作用是通过其受体PRLr介导的。PRL的作用是刺激人类乳腺癌的生长、运动和进展。配体结合后，Nek3丝氨酸/苏氨酸激酶和Vav2鸟嘌呤核苷酸交换因子（GEF）的复合物与PRLr结合。我们的实验室已经证明Nek3是Vav2磷酸化和GEF活性所必需的，并且在恶性（与正常）乳腺组织中高度过表达。由于sirna介导的Nek3敲低，prl诱导的丝状肌动蛋白的形成、运动和乳腺癌细胞的侵袭被抑制。为了进一步将这些体外研究结果与体内生物学联系起来，我们的实验室最近产生了一只基因Nek3-/-小鼠。Nek3-/-小鼠表现出明显的乳腺表型，末端芽减弱，分支改变，上皮细胞祖细胞显著缺失。为了更好地了解Nek3/Vav2复合体在乳腺癌发病过程中的功能，我们的实验室试图通过靶向蛋白质组学分析确定该复合体的下游底物。这些研究证实了prl诱导的Nek3/Vav2与局灶黏附蛋白paxillin以及转录因子Stat5和NFAT的关联。事实上，我们的分析表明Nek3调控paxillin磷酸化，Nek3/Vav2复合物结合并修饰prl调控基因的染色质，调节NFAT和Stat5的活性。因此，Nek3/Vav2复合体协调调节paxillin、Stat5和NFAT的功能是本研究的中心假设，这些蛋白都与乳腺癌的发病机制有关。这一假设将在三个特定的目标中进行测试，使用具有代表性的乳腺癌线和模型。首先，将通过诱变和磷酸化特异性抗体鉴定prl诱导的nek3介导的paxillin丝氨酸磷酸化位点，并鉴定其功能意义。其次，将评估Nek3/Vav2复合物对Stat5-和nfat介导的基因表达的核内动力学和调控。第三，Nek3/Vav2复合物的体内生物学相关性将通过Nek3-/-小鼠和Nek3活性水平改变的乳腺癌细胞异种移植物的研究来解决。公共卫生相关性：拟就Nek3/Vav2复合体在人类乳腺癌中的研究具有高度相关性，因为它们将详细说明这种在乳腺癌细胞中过表达的复合体如何调节癌细胞转移和基因表达。此外，这些研究将为开发和测试Nek3/Vav2复合物的靶向抑制剂治疗该疾病提供基础。

DESCRIPTION (provided by applicant): The actions of prolactin (PRL), a hormone utilized in normal and malignant mammary tissues, are mediated through its receptor, the PRLr. PRL acts to stimulate the growth, motility, and progression of human breast cancer. Following binding of ligand, a complex of the Nek3 serine/threonine kinase and the Vav2 guanine nucleotide exchange factor (GEF) associates with the PRLr. Our lab has demonstrated that Nek3 is required for Vav2 phosphorylation and GEF activity and is highly overexpressed in malignant (vs. normal) breast tissues. As a consequence of SiRNA-mediated Nek3 knockdown, the PRL-induced formation of filamentous actin, motility, and invasion of breast cancer cells was inhibited. To further correlate these in vitro findings with in vivo biology, a congenic Nek3-/- mouse was recently generated for our lab. The Nek3-/- mouse demonstrates a distinct mammary phenotype of attenuated terminal end buds and altered branching, and demonstrates a significant loss of epithelial cell progenitors. To better understand the function of the Nek3/Vav2 complex during the pathogenesis of breast cancer our lab has sought to identify downstream substrates of this complex through targeted proteomic analysis. These studies have demonstrated a PRL-induced association of the Nek3/Vav2 with the focal adhesion protein paxillin and the transcription factors Stat5 and NFAT. Indeed, our analysis demonstrates that Nek3 regulates paxillin phosphorylation and that the Nek3/Vav2 complex associates with and modifies the the chromatin of PRL-regulated genes, regulating the activity of NFAT and Stat5. It is the central hypothesis, therefore, of this proposal that the Nek3/Vav2 complex coordinately regulates the function of paxillin, Stat5, and NFAT, proteins all implicated in the pathogenesis of breast cancer. This hypothesis will be tested in three specific aims using representative breast cancer lines and models. First, the sites of PRL-induced, Nek3-mediated serine phosphorylation on paxillin will be identified and characterized as to functional significance through the use of mutagenesis and phospho-specific antibodies. Second, the intranuclear dynamics and regulation of the Stat5- and NFAT-mediated gene expression by the Nek3/Vav2 complex will be assessed. Third, the in vivo biologic relevance of the Nek3/Vav2 complex will be addressed through the study of Nek3-/- mice and breast cancer cell xenografts with altered levels of Nek3 activity. PUBLIC HEALTH RELEVANCE: The proposed studies on the Nek3/Vav2 complex in human breast cancer are highly relevant as they will detail how this complex, that is overexpressed in a breast cancer cell, regulates cancer cell metastasis and gene expression. In addition, these studies will provide a basis for the development and testing of targeted inhibitors of the Nek3/Vav2 complex in the treatment of this disease.