Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor.
The interactions of H-Ras, R-Ras, and Rap1A with the Ras-binding domains (RBD) of the c-Raf kinase and of the Ral guanine nucleotide exchange factor (RGF) was studied biochemically in solution. From deletion cloning the RGF-RBD was defined as a 97-amino acid-long fragment from the C-terminal end of the human RGF, which is an independent folding domain with high stability. Interestingly, whereas H-Ras binds with high affinity (KD = 20 nM) to Raf-RBD and with low affinity (KD = 1 microM) to RGF-RBD, Rap1A shows the opposite behavior. The binding of both RBDs to R-Ras is weak and shows no specificity. The interaction between Rap1A and RGF-RBD shows similar characteristics to the Ras-Raf interaction because it is blocked by mutations in the effector region (D38A) and it inhibits the dissociation of guanine nucleotide, which is the basis for the quantitative measurements in this work. Furthermore, the binding of RGF-RBD inhibits the interaction between Rap1A and Rap-GAP. As long as the cellular localizations of the different proteins and their biological functions are not clarified, these biochemical data seem to indicate that Ral-guanine nucleotide exchange factors is an effector molecule of Rap1A rather than of H-Ras.
Herrmann
C
C
Max-Planck-Institut für Molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany.
Horn
G
G
Spaargaren
M
M
Wittinghofer
A
A
D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D020725:ral GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D020663:ras Guanine Nucleotide Exchange Factors
D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019204:GTP-Binding Proteins; D020725:ral GTP-Binding Proteins; D020668:rap GTP-Binding Proteins