Sec14 Domain-Mediated Regulation of the Dbs Oncogene

Sec14 域介导的 Dbs 癌基因调控

• 批准号: 6885510
• 负责人: ELENA V KOSTENKO
• 金额: $ 4.99万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2007-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6885510
• 关键词: 3T3 cells; binding proteins; biological signal transduction; carcinogenesis; gene mutation; genetic regulation; green fluorescent proteins; guanine nucleotide exchange factors; immunofluorescence technique; immunoprecipitation; lipids; molecular /cellular imaging; oncogenes; oncoproteins; protein protein interaction; protooncogene

DESCRIPTION (provided by applicant): The high transforming potential of constitutively activated Rho specific guanine nucleotide exchange factors (RhoGEFs) suggests an important role for the RhoGEF family members in oncogenic transformation. Consistent with this, BCR and LARG are already known to be contributing partners to chromosomal rearrangements that are associated with specific human leukemias (CML and MLL respectively). The Dbs oncogene encodes a RhoGEF that has potent transforming activity in both fibroblasts and human breast epithelial cells. We have determined that the transforming activity and intracellular localization of Dbs is regulated by an NH2-terminal Sec14 homology lipid binding domain, which is also found in several other family members (Kalirin, Dbl, Trio, UNC-73). The aim of this proposal is to determine the molecular mechanism of Sec14 domain-mediated regulation of Dbs transforming activity. Using a homology model of this domain, in combination with liposome binding assays, we will identify mutations that completely disrupt lipid binding by this domain. We will then use these mutants to determine the role of lipid binding by the Sec14 domain in the regulation of proto-Dbs subcellular localization and transforming activity. Additionally, we will test if the Sec14 domain of Dbs can directly bind to its PH domain, and investigate the respective contributions of the Sec14 domain-mediated lipid binding, and PH domain binding, to Dbs transformation. Finally, we will use an immunofluorescence approach to visualize and compare spatio-specific RhoA activation by proto and onco-Dbs proteins. If our working model for Sec14 domain-mediated Dbs regulation is correct, this could establish a new paradigm for RhoGEF regulation.

描述（由申请人提供）：组成型激活的 Rho 特异性鸟嘌呤核苷酸交换因子 (RhoGEF) 的高转化潜力表明 RhoGEF 家族成员在致癌转化中的重要作用。与此一致，已知 BCR 和 LARG 是与特定人类白血病（分别为 CML 和 MLL）相关的染色体重排的贡献伙伴。 Dbs 癌基因编码的 RhoGEF 在成纤维细胞和人乳腺上皮细胞中具有有效的转化活性。我们已经确定 Dbs 的转化活性和细胞内定位是由 NH2 末端 Sec14 同源脂质结合结构域调节的，该结构域也存在于其他几个家族成员（Kalirin、Dbl、Trio、UNC-73）中。本提案的目的是确定 Sec14 结构域介导的 Dbs 转化活性调节的分子机制。使用该结构域的同源模型，结合脂质体结合测定，我们将鉴定完全破坏该结构域的脂质结合的突变。然后，我们将使用这些突变体来确定 Sec14 结构域的脂质结合在调节原 Dbs 亚细胞定位和转化活性中的作用。此外，我们将测试Dbs的Sec14结构域是否可以直接与其PH结构域结合，并研究Sec14结构域介导的脂质结合和PH结构域结合对Dbs转化的各自贡献。最后，我们将使用免疫荧光方法来可视化和比较 proto 和 onco-Dbs 蛋白的空间特异性 RhoA 激活。如果我们的 Sec14 域介导的 Dbs 调节工作模型是正确的，这可能会为 RhoGEF 调节建立一个新的范例。