Molecular Regulation of the Protooncogene Dbl

Dbl在1985年被鉴定为一种新的癌基因，已成为Rho家族小GTP酶的鸟嘌呤核苷酸交换因子（GEF）的扩展家族的原型。GEF通过催化结合的GDP交换为GTP来激活小G蛋白。这种在G蛋白调节中的重要作用，以及对许多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的理解有限。在理解一些Rho GEF的调节方面已经取得了进展，但是关于dbl原癌基因（proto-dbl）的体内调节知之甚少。以前的实验表明，从115 kDa的原Dbl的前497个氨基酸的截短产生了66 kDa的癌蛋白，其转化活性为50-70倍。这一发现表明，497个氨基酸的N-末端序列在原Dbl的鸟嘌呤核苷酸交换和转化活性的调节中起着重要作用。本研究的目的是阐明调节原Dbl的分子机制。我们将确定：（1）下调转化的N-末端结构域内的特定区域及其在靶向蛋白质的亚细胞定位中的作用，（2）GEF活性的抑制是否是通过分子内或分子间机制，并鉴定与原Db 1的N-末端区域相互作用的分子，（3）与GEF、Fgd 1相比，原Db 1的N端调控机制是否具有DH/PH结构域的特异性。

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.