Molecular Regulation of the Protooncogene Dbl

Dbl于1985年被确定为一种新的致癌基因，现已成为小gtpase Rho家族的鸟嘌呤核苷酸交换因子（gef）家族的原型。gef通过催化结合的GDP交换GTP来激活小g蛋白。这种在g蛋白调控中的重要作用，以及许多gef引起细胞转化的认识，引起了人们对这些蛋白质的极大兴趣。虽然许多研究都集中在Dbl家族gef通过与Rho家族蛋白相互作用诱导转化的机制上，但对上游刺激通过这些关键的小g蛋白传递信息的机制了解有限。在了解一些Rho gef的调控方面已经取得了进展，但对dbl原癌基因（proto-dbl）的体内调控知之甚少。先前的实验表明，从115 kDa的原dbl中截断前497个氨基酸产生66 kDa的癌蛋白，其转化活性是其50-70倍。这一发现表明497个氨基酸的n端序列在调节原dbl的鸟嘌呤核苷酸交换和转化活性中起重要作用。本研究的目的是阐明调节原dbl的分子机制。我们将确定：(1)在n端域中下调转化的特定区域及其在靶向蛋白质亚细胞定位中的作用，(2)GEF活性的抑制是否通过分子内或分子间机制，并鉴定与原- dbl的n端区域相互作用的分子，以及(3)与GEF Fgd1相比，原- dbl的n端调节机制是否特异性针对原- dbl的DH/PH结构域。

Identified in 1985 as a novel oncogene, Dbl has become the prototype of an expanding family of guanine nucleotide exchange factors (GEFs) for the Rho family of small GTPases. GEFs activate small G-proteins by catalyzing the exchange of bound GDP for GTP. This important role in G-protein regulation, and the recognition that many GEFs cause cellular transformation, has generated great interest in these proteins. While much research has focused on the mechanisms by which Dbl family GEFs induce transformation through interactions with Rho family proteins, there is limited understanding of the mechanisms by which upstream stimuli transmit messages via these critical small G-proteins. Progress has been made in understanding the regulation of a few Rho GEFs, but little is known about the in vivo regulation of the dbl proto- oncogene (proto-dbl). Previous experiments revealed that truncation of the first 497 amino acids from the 115 kDa proto-Dbl resulted in a 66 kDa oncoprotein with 50-70 times its transforming activity. This finding suggests that the 497 amino acid N-terminal sequence plays an important role in the regulation of the guanine nucleotide exchange and transforming activities of proto-Dbl. The goal of this study is to elucidate the molecular mechanisms which regulate proto-Dbl. We will determine: (1) the specific region within the N-terminal domain which down- regulates transformation and its role in targeting the subcellular localization of the protein, (2) whether inhibition of GEF activity is through an intra- or intermolecular mechanism and identify molecules that interact with the N-terminal region of proto-Dbl, and (3) whether the mechanisms of N-terminal regulation of proto-Dbl are specific for the DH/PH domains of proto-Dbl by comparison with the GEF, Fgd1.