Multimeric Signaling Complexes in PRLr Transduction

催乳素(PRL)是一种在正常和恶性乳腺组织中使用的激素，其作用如下 通过其受体PRLr介导。催乳素作用于刺激人类的生长、运动和进步 乳腺癌。在配体结合后，Nek3丝氨酸/苏氨酸激酶与Vav2的复合体 鸟嘌呤核苷酸交换因子与PRLr密切相关。我们的实验室已经证明Nek3是 是Vav2磷酸化和全球环境生长因子活性所必需的，在恶性(与正常)乳腺组织中高度过度表达 纸巾。作为siRNA介导的Nek3基因敲除的结果，PRL诱导的丝状细胞的形成 乳腺癌细胞的肌动蛋白、运动能力和侵袭能力受到抑制。为了进一步将这些体外研究结果与 在活体生物学方面，最近为我们的实验室培育了一个同源基因的nek3/-小鼠。Nek3-/-鼠标演示 一种独特的乳房表型，末端芽变薄，分支改变，表现为 上皮祖细胞显著丧失。为了更好地了解Nek3/Vav2复合体在 乳腺癌的发病机制我们的实验室试图确定这种复合体的下游底物 通过有针对性的蛋白质组分析。这些研究已经证明了催乳素诱导的 Nek3/Vav2与粘着斑蛋白Paxlin及转录因子Stat5和NFAT结合。的确，我们的 分析表明，Nek3调节帕克西林的磷酸化，Nek3/Vav2复合体 结合并修饰PRL调节基因的染色质，调节NFAT和 统计数据5.因此，这一提议的中心假设是Nek3/Vav2复合体是协调的 调节Paxlin、Stat5和NFAT的功能，这些蛋白都与乳腺癌的发病机制有关。 这一假说将使用具有代表性的乳腺癌株系和模型在三个特定目标上进行测试。第一, 将鉴定和表征PRL诱导的、nek3介导的帕西林上的丝氨酸磷酸化的位置。 至于通过使用突变和磷酸特异性抗体的功能意义。第二， Nek3/Vav2对Stat5和NFAT介导的基因表达的核内动力学调控 综合体将被评估。第三，Nek3/Vav2复合体的体内生物学相关性将被解决 通过对Nek3-/-小鼠和乳腺癌细胞异种移植的研究，发现Nek3活性水平发生了变化。

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.