Multimeric Signaling Complexes in PRLr Transduction

PRLr 转导中的多聚信号复合物

• 批准号: 6949853
• 负责人: Charles V Clevenger
• 金额: $ 4.48万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-13 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6949853
• 关键词: RNA; biological signal transduction; breast neoplasms; cell line; cell membrane; cell motility; chemoattractants; epithelium; guanine nucleotide exchange factors; heterozygote; hormone receptor; hybrid cells; immunoprecipitation; integrins; intracellular; ligands; mammary gland; mass spectrometry; microtubule associated protein; mutant; phenotype; phenylalanine; phosphorylation; prolactin; protein kinase; stoichiometry; transfection; tyrosine; ultracentrifugation; yeasts

The actions of prolactin (PRL), a hormone utilized in normal and malignant mammary tissues, are mediated through its receptor (PRLr), a member of the cytokine receptor superfamily. PRL acts to stimulate the survival, motility, and the progression of human breast cancer. PRL-induced dimerization of the PRLr induces the association, tyrosine phosphorylation, and activation of members of the Vav family (Vav1-3) of guanine nucleotide exchange factors (GEF), an event necessary for PRL-driven cellular signaling and function. We have discovered two novel protein kinases in association with Vav1 and Vav2, namely the tyrosine kinase Tec, and the serine/threonine kinase Nek3. Both kinases are rapidly activated during PRL-stimulated signaling and associate with the PRLr-Vav2 complex in human breast cancer, forming a macromolecular signaling assembly. It is the central hypothesis of this proposal that the structures and interactions in the PRLr-Tec/Vav2/Nek3 complex coordinately activate the downstream effectors and functions associated with PRLr action. This hypothesis will be tested by three specific aims using breast cancer and PRL-responsive lines. First, the functional role of Tec and Nek3 during PRL-driven proliferation, survival, and cytoskeletal change will be assessed at the cellular and molecular levels, through the transfection of wild-type, activated, and kinase-dead forms of Tec and Nek3. Second, the location of Vav2 tyrosine phosphorylation will be determined by mass spectroscopy and the functional significance of these residues evaluated by replacement mutagenesis. Third, mutagenic and biophysical approaches will identify and characterize the interaction motifs within the PRLr-Tec/Vav2 complex, while the role of such motifs during PRLr-mediated signaling and action will be evaluated through the transfection of interaction defective mutants of the PRLr, Tec and Vav2. The structure/function studies proposed here will provide insight into the assembly of a multimeric PRLr-associated signaling complex and relate these findings to cellular processes of relevance to the pathophysiology of human breast cancer. These analyses of PRLr-associated transduction may ultimately provide the basis for novel therapeutic strategies aimed at modulating the function of the PRL/PRLr complex.

泌乳素（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，PRLr是细胞因子受体超家族的成员。PRL的作用是促进人类乳腺癌的生存、运动和进展。prl诱导的PRLr二聚化诱导了鸟嘌呤核苷酸交换因子（GEF）的关联、酪氨酸磷酸化和Vav家族成员（Vav1-3）的激活，这是prl驱动的细胞信号传导和功能所必需的事件。我们发现了两种与Vav1和Vav2相关的新型蛋白激酶，即酪氨酸激酶Tec和丝氨酸/苏氨酸激酶Nek3。这两种激酶在prl刺激的信号传导过程中被迅速激活，并与人类乳腺癌中的PRLr-Vav2复合物相关联，形成一个大分子信号传导组装体。该研究的核心假设是，PRLr- tec /Vav2/Nek3复合体的结构和相互作用协同激活与PRLr作用相关的下游效应物和功能。这一假设将通过使用乳腺癌和prl应答细胞系的三个特定目标进行检验。首先，Tec和Nek3在prl驱动的增殖、存活和细胞骨架变化过程中的功能作用将在细胞和分子水平上进行评估，通过转染野生型、激活型和激酶死亡形式的Tec和Nek3。其次，将通过质谱确定Vav2酪氨酸磷酸化的位置，并通过替代诱变评估这些残基的功能意义。第三，诱变和生物物理方法将识别和表征PRLr-Tec/Vav2复合体中的相互作用基序，而这些基序在PRLr介导的信号传导和作用中的作用将通过转染PRLr、Tec和Vav2的相互作用缺陷突变体来评估。本文提出的结构/功能研究将深入了解多聚prlr相关信号复合物的组装，并将这些发现与与人类乳腺癌病理生理相关的细胞过程联系起来。这些对PRLr相关转导的分析可能最终为旨在调节PRL/PRLr复合物功能的新治疗策略提供基础。