PDGF Receptor Regulation by CBL

描述(申请人提供)：PDGF受体酪氨酸激酶在发育过程中的细胞增殖、迁移和分化、伤口愈合和组织重建中发挥关键的生理作用。这些受体的激活与细胞增殖有关；人类癌症，特别是胶质瘤的侵袭性和血管生成，以及PDGFR融合癌基因导致了髓系白血病的一个亚群。事实上，PDGFR激酶活性的抑制剂正在被评估为抗癌药物。因此，了解调节PDGF受体下游信号水平的机制是细胞和癌症生物学研究的主要目标。配体诱导的下调，代表了溶酶体降解和再循环到细胞表面之间的平衡，构成了受体酪氨酸激酶信号效力的主要决定因素。我们最近的工作已经确定Cbl原癌蛋白是PDGF受体下调的关键调节因子。CBL是一种泛素连接酶，它针对激活的PDGF受体进行泛素化，进而促进其溶酶体的分选。依赖Cbl的泛素化如何作为PDGF受体的溶酶体分选信号尚不清楚。基于最近的酵母遗传学研究，我们假设了一种新的内体分选复合体ESCRT-1，它整合了TSG101肿瘤抑制蛋白，在依赖Cbl和泛素的PDGF受体溶酶体分选中发挥了作用。在这里，我们将使用一些互补的方法来检验这一假设。我们将使用Cbl缺乏的小鼠胚胎成纤维细胞、在PDGFR缺失的成纤维细胞中表达的Cbl不敏感的PDGFR突变体、泛素化条件缺陷的细胞以及PDGFR-泛素融合蛋白来确定依赖Cbl的泛素化在PDGFR下调中的重要作用。我们将使用双重免疫标记研究来确定依赖Cbl的PDGFR溶酶体分选发生的间隔(S)。我们将使用生化和共定位分析来确定PDGFR和ESCRT-1相互作用，并使用过度表达和显性-负向方法来确定这种新的蛋白质复合体在PDGFR溶酶体分选中的要求。功能分析将评估ESCRT-1复合体是否在Cb1介导的PDGFR信号下调中起关键作用。通过这些研究获得的见解应该会加强对一个基本的细胞过程的分子理解，即下调激活的受体酪氨酸激酶。鉴于PDGF受体在恶性细胞增殖、侵袭和血管生成中的作用，我们的研究与癌症特别相关。识别受体酪氨酸激酶功能的新调节机制可能为开发新的治疗药物和/或化学和放射增敏剂提供途径。

DESCRIPTION (provided by applicant): PDGF receptor tyrosine kinases play crucial physiological roles in cell proliferation, migration and differentiation during development, wound healing and tissue remodeling. Activation of these receptors is linked to cell proliferation; invasiveness and angiogenesis in human cancers, in particular glial tumors, and PDGFR fusion oncogenes are responsible for a subset of myelogenous leukemia. Indeed, inhibitors of PDGFR kinase activity are being evaluated as anti-cancer agents. Understanding the mechanisms that regulate the level of signaling downstream of PDGF receptors is therefore a major goal of research in cell and cancer biology. Ligand-induced downregulation, representing a balance between lysosomal degradation and recycling to cell surface, constitutes a major determinant of signaling potency of receptor tyrosine kinases. Our recent work has established the Cbl proto-oncoprotein as a crucial regulator of PDGF receptor down-regulation. Cbl, an ubiquitin ligase, targets activated PDGF receptors for ubiquitination that in turn facilitates their lysosomal sorting. How Cbl-dependent ubiquitination functions as a lysosomal sorting signal for PDGF receptors is unknown. Based on recent yeast genetic studies, we hypothesize the role of a novel endosomal sorting complex, ESCRT-1, which incorporates the TSG101 tumor suppressor protein, in Cbl- and ubiquitin-dependent lysosomal sorting of PDGF receptor. Here, we will test this hypothesis using a number of complementary approaches. We will use Cbl-deficient mouse embryonic fibroblasts, Cbl-insensitive PDGFR mutants expressed in PDGFR-null fibroblasts, cells with a conditional defect in ubiquitination, and PDGFR-ubiquitin fusion proteins to establish the essential role of Cbl-dependent ubiquitination in PDGFR down-regulation. We will use dual immunolabeling studies to identify the compartment(s) where Cbl-dependent lysosomal sorting of PDGFR occurs. We will use biochemical and co-localization analyses to establish that PDGFR and ESCRT-1 interact, and use over-expression and dominant-negative approaches to establish the requirement of this novel protein complex in lysosomal sorting of PDGFR. Functional analyses will assess if ESCRT-1 complex is critical in Cbl-mediated downregulation of PDGFR signaling. Insights gained through these studies should enhance the molecular understanding of a basic cellular process, downregulation of activated receptor tyrosine kinases. Given the role of PDGF receptors in maligant cell proliferation, invasiveness and angiogenesis, our studies are particularly relevant to cancer. Identification of novel regulatory mechanisms of receptor tyrosine kinase function may provide avenues to develop newer therapeutic agents and/or chemo- and radio-sensitizers.