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 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.
Wolthuis
R M
RM
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
Bauer
B
B
van 't Veer
L J
LJ
de Vries-Smits
A M
AM
Cool
R H
RH
Spaargaren
M
M
Wittinghofer
A
A
Burgering
B M
BM
Bos
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020726:ral Guanine Nucleotide Exchange Factor; D020668:rap GTP-Binding Proteins; D018631:ras Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020666:ras-GRF1
C542351:CDC25 protein, S cerevisiae; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; C490771:RGL1 protein, human; C097104:Rgl1 protein, mouse; C490805:Rgl2 protein, mouse; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D020726:ral Guanine Nucleotide Exchange Factor; D020666:ras-GRF1; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D018631:ras Proteins