RalGDS-like factor (Rlf) is a novel Ras and Rap 1A-associating protein

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 1A(Val12) but not with Rap 1A(Asn17). 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 μM and 0.4 μM, 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.