PDGF Receptor Regulation by CBL

描述（由申请方提供）：PDGF受体酪氨酸激酶在发育、伤口愈合和组织重塑期间的细胞增殖、迁移和分化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，特别是神经胶质瘤中的侵袭性和血管生成，以及PDGFR融合癌基因负责髓性白血病的一个子集。事实上，PDGFR激酶活性的抑制剂正在被评估为抗癌剂。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转导效力的主要决定因素。我们最近的工作已经确定了Cbl原癌蛋白作为PDGF受体下调的关键调节因子。Cbl是一种泛素连接酶，靶向活化的PDGF受体进行泛素化，进而促进其溶酶体分选。Cbl依赖的泛素化如何作为PDGF受体的溶酶体分选信号是未知的。基于最近的酵母遗传学研究，我们假设的作用，一种新的内体分选复合物，ESCRT-1，其中包括TSG 101肿瘤抑制蛋白，在Cbl和泛素依赖性的溶酶体分选的PDGF受体。在这里，我们将使用一些互补的方法来测试这个假设。我们将使用Cbl缺陷的小鼠胚胎成纤维细胞，Cbl不敏感的PDGFR突变体表达的PDGFR无效的成纤维细胞，细胞与条件缺陷的泛素化，和PDGFR-泛素融合蛋白，以建立Cbl依赖的泛素化在PDGFR下调的重要作用。我们将使用双重免疫标记研究来鉴定发生PDGFR的Cbl依赖性溶酶体分选的隔室。我们将使用生物化学和共定位分析来确定PDGFR和ESCRT-1相互作用，并使用过表达和显性负性方法来确定这种新型蛋白质复合物在PDGFR溶酶体分选中的需求。功能分析将评估ESCRT-1复合物是否在Cbl介导的PDGFR信号转导下调中至关重要。通过这些研究获得的见解应加强对基本细胞过程的分子理解，即激活的受体酪氨酸激酶的下调。鉴于PDGF受体在恶性细胞增殖、侵袭性和血管生成中的作用，我们的研究与癌症特别相关。受体酪氨酸激酶功能的新的调节机制的鉴定可能提供开发新的治疗剂和/或化学和放射增敏剂的途径。

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.