Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Base Sequence
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cloning, Molecular
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DNA, Complementary / genetics*
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DNA, Complementary / isolation & purification*
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Fungal Proteins / genetics
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Fungal Proteins / metabolism
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GTP-Binding Proteins / genetics*
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GTP-Binding Proteins / isolation & purification*
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GTP-Binding Proteins / metabolism
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Guanine Nucleotide Exchange Factors*
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Guanosine Triphosphate / metabolism
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Mice
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Molecular Sequence Data
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Protein Binding
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins
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Sequence Homology, Amino Acid
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Transcription Factors / genetics*
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Transcription Factors / isolation & purification*
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Transcription Factors / metabolism
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ral Guanine Nucleotide Exchange Factor
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rap GTP-Binding Proteins
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ras Proteins / genetics*
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ras Proteins / isolation & purification*
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ras Proteins / metabolism
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ras-GRF1*
Substances
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CDC25 protein, S cerevisiae
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Cell Cycle Proteins
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DNA, Complementary
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Fungal Proteins
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Guanine Nucleotide Exchange Factors
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RGL1 protein, human
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Rgl1 protein, mouse
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Rgl2 protein, mouse
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Saccharomyces cerevisiae Proteins
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Transcription Factors
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ral Guanine Nucleotide Exchange Factor
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ras-GRF1
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Guanosine Triphosphate
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GTP-Binding Proteins
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rap GTP-Binding Proteins
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ras Proteins
Associated data
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GENBANK/T08669
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GENBANK/U54639