Wolthuis R M, Bauer B, van 't Veer L J, de Vries-Smits A M, Cool R H, Spaargaren M, Wittinghofer A, Burgering B M, Bos J L
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
Oncogene. 1996 Jul 18;13(2):353-62.
The small GTPase Rap 1A is a close relative of Ras that, when overexpressed, is able to revert oncogenic transformation induced by active Ras. We screened a mouse embryonic cDNA library using the yeast two-hybrid system and isolated the cDNA of a novel Rap 1A-interacting protein. The open reading frame encodes for an 84 kDa protein with a Cdc25-homology domain which shares approximately 30% identity with Ral guanine nucleotide dissociation stimulator (RalGDS) and RalGDS-like (Rg1). The C-terminal region reveals a striking conservation of sequences with the Ras-binding domain of RalGDS. We designated this protein Rlf, for RalGDS-like factor. In the yeast system, Rlf interacts with Rap 1A, H-Ras and R-Ras, but not with Rac and Rho. In addition, we found that Rlf interacts with Rap 1Aval12 but not with Rap 1AAsn17. In vitro binding studies revealed that a C-terminally located 91 amino acid region of Rlf is sufficient for direct association with the GTP-bound form of Ras and Rap 1A. The observed dissociation constants are 0.6 microM and 0.4 microM, respectively. No significant association with Ras-GDP or Rap 1A-GDP could be detected. These binding characteristics indicate that Rlf is a putative effector for Ras and Rap 1A.
小GTP酶Rap 1A是Ras的近亲，当过度表达时，它能够逆转由活性Ras诱导的致癌转化。我们使用酵母双杂交系统筛选了小鼠胚胎cDNA文库，并分离出一种新型Rap 1A相互作用蛋白的cDNA。开放阅读框编码一种84 kDa的蛋白，该蛋白具有一个Cdc25同源结构域，与Ral鸟嘌呤核苷酸解离刺激因子（RalGDS）和RalGDS样蛋白（Rg1）具有约30%的同一性。C末端区域与RalGDS的Ras结合结构域具有显著的序列保守性。我们将这种蛋白命名为Rlf，即RalGDS样因子。在酵母系统中，Rlf与Rap 1A、H-Ras和R-Ras相互作用，但不与Rac和Rho相互作用。此外，我们发现Rlf与Rap 1Aval12相互作用，但不与Rap 1AAsn17相互作用。体外结合研究表明，Rlf位于C末端的91个氨基酸区域足以与GTP结合形式的Ras和Rap 1A直接结合。观察到的解离常数分别为0.6 microM和0.4 microM。未检测到与Ras-GDP或Rap 1A-GDP的显著结合。这些结合特性表明Rlf是Ras和Rap 1A的一种假定效应器。
