Multimeric Signaling Complexes in PRLr Transduction

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，该受体是细胞因子受体超家族的成员。PRL的作用是刺激人乳腺癌的存活、运动和进展。 PRL诱导的PRLr二聚化诱导鸟嘌呤核苷酸交换因子（GEF）的Vav家族（Vav 1 -3）成员的缔合、酪氨酸磷酸化和活化，这是PRL驱动的细胞信号传导和功能所必需的事件。 我们已经发现了两个新的蛋白激酶与Vav 1和Vav 2，即酪氨酸激酶Tec，和丝氨酸/苏氨酸激酶Nek 3。 这两种激酶在PRL刺激的信号传导过程中被快速激活，并与人乳腺癌中的PRLr-Vav 2复合物结合，形成大分子信号传导组装。 该提议的中心假设是PRLr-Tec/Vav 2/Nek 3复合物中的结构和相互作用协调激活与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反应株系的三个特定目标进行检验。 首先，Tec和Nek 3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通过转染野生型，活化和激酶死亡形式的Tec和Nek 3进行评估。 其次，Vav 2酪氨酸磷酸化的位置将通过质谱法确定，并且通过置换诱变评价这些残基的功能意义。 第三，诱变和生物物理学方法将鉴定和表征PRLr-Tec/Vav 2复合物内的相互作用基序，而这些基序在PRLr介导的信号传导和作用期间的作用将通过转染PRLr、Tec和Vav 2的相互作用缺陷突变体来评估。 这里提出的结构/功能的研究将提供深入了解组装的多聚体PRL受体相关的信号传导复合物，并将这些发现与人类乳腺癌的病理生理相关的细胞过程。 这些分析的催乳素相关的转导可能最终提供了新的治疗策略，旨在调节PRL/PRLr复合物的功能的基础。

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.