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.
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