ERBB RECEPTOR CONTROL OF BREAST CANCER GROWTH

表皮生长因子受体(ErbB)家族的重要作用 在乳腺癌的发展中的成员已经被证明超过 过去的15年里。我们最近克隆了一种新的蛋白质EBP1，它 与erbB3的胞质结构域相互作用。对细胞的处理 ErbB-3配体HERG诱导EBP1易位 从细胞质到细胞核。EBP1的过表达抑制 人乳腺癌细胞的增殖。我们建议研究 Erb B-3EBP1相互作用在人乳腺癌中的生理功能 细胞。了解这些规则及其后果 相互作用可能导致对erbB-3信号的更好理解 转导和乳房新治疗靶点的定义 癌症。 首先，为了定义erbB-3-EBP1相互作用的功能，我们将a) 确定HRG介导的EBP1与其他erb B受体的相互作用 以及这种相互作用对erbB-3表达的依赖性 使用酵母-双杂交系统的异二聚体结合伴侣和 表达erb B特定组合的工程化小鼠细胞 受体2)共聚焦建立EBP1和erbB-3的共定位 HRG治疗前后的显微镜检查，c)确定 Erb B-3结合所需的EBP1及其表达的影响 EBP1的不同区域在HRG介导的信号转导中的作用 EBP1，d)的缺失和单点突变决定了 EBP1过表达在基础和HRG诱导的细胞生长和 利用稳定的、可诱导表达的转染体进行分化。 第二，我们将确定蛋白激酶C(PKC)在 调节EBP1的活性。心率变异性后的磷酸化部位 治疗将使用胰酶磷酸肽图和 磷酸分析。蛋白激酶C抑制剂对EBP1功能的影响 将在完整的细胞中进行评估。潜在的PKC位点将发生突变 以及这些突变对EBP1功能的影响。第三，我们将 明确EBP1核定位的意义。我们将a) 确定EBP1是否是多蛋白核定位复合体的一部分 利用酵母双杂交系统分离EBP1相互作用蛋白 B)确定EBP1及其辅助转录激活因子的作用 使用GAL4 DNA结合域融合c)识别首选的DNA结合 使用简并寡核苷酸策略的EBP1的位点。

A crucial role for Epidermal Growth Factor Receptor (erbB) family members in development of breast cancer has been demonstrated over the past 15 years. We have recently cloned a novel protein, EBP1, which interacts with the cytoplasmic domain of erbB3. Treatment of cells with the erbB-3 ligand, heregulin (HRG) induces the translocation of EBP1 from the cytoplasm to the nucleus. Overexpression of EBP1 inhibits proliferation of human breast cancer cells. We propose to study the physiological function of erbB-3EBP1 interactions in human breast cancer cells. Understanding the regulation and consequences of these interactions could lead to a better understanding of erbB-3 signal transduction and to the definition of new therapeutic targets in breast cancer. First, to define the function of erbB-3-EBP1 interactions, we will a) determine HRG-mediated interactions of EBP1 with other erbB receptors and the dependence of such interactions on the expression of erbB-3 heterodimeric binding partners using the yeast-two hybrid system and engineered murine cells expressing defined combinations of erbB receptors 2) establish the colocalization of EBP1 and erbB-3 by confocal microscopy before and after HRG treatment, c) determine the region of EBP1 required for erbB-3 binding and the effects of expression of different regions of EBP1 on HRG-mediated signalling using a series of deletion and single site mutants of EBP1, d) determine the effects of EBP1 overexpression on basal and HRG-induced cell growth and differentiation using stable transfectants with inducible expression. Second, we will determine the role of Protein Kinase C (PKC) in modulating EBP1 activity. The sites of phosphorylation after HRG treatment will be determined using tryptic phosphopeptide mapping and phosphamino acid analysis. Effects of PKC inhibitors on EBP1 function will be assessed in intact cells. Potential PKC sites will be mutated and effects of such mutations on EBP1 function defined. Third, we will define the significance of EBP1 nuclear localization. We will a) determine if EBP1 is part of a multiprotein nuclear localization complex by isolating EBP1 interacting proteins using the yeast two-hybrid system b) define the role of EBP1 and its accessory transcriptional activators using GAL4 DNA binding domain fusions c) identify preferred DNA binding sites for EBP1 using a degenerate oligonucleotide strategy.