Sec14 Domain-Mediated Regulation of the Dbs Oncogene

描述（由申请人提供）：组成激活的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起重要作用。与此一致的是，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合作伙伴。Dbs致癌基因编码的RhoGEF在成纤维细胞和人乳腺上皮细胞中都具有强大的转化活性。我们已经确定Dbs的转化活性和细胞内定位受nh2末端Sec14同源脂质结合结构域的调节，这也在其他几个家族成员（Kalirin, Dbl, Trio, UNC-73）中发现。本提案的目的是确定Sec14结构域介导的Dbs转化活性调控的分子机制。利用该结构域的同源性模型，结合脂质体结合试验，我们将确定完全破坏该结构域脂质结合的突变。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蛋白对RhoA的空间特异性激活。如果我们对Sec14结构域介导的Dbs调控的工作模型是正确的，这可能为RhoGEF调控建立一个新的范式。

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.