Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase.
The Ras-binding domain (RBD) of human Raf-1 was purified from Escherichia coli, and its interaction with Ras was investigated. Its dissociation constant with p21ras.guanyl-5'-yl imidodiphosphate was found to be 18 nM, with a slight preference for H-ras over K- and N-ras. Oncogenic forms bind with slightly lower affinity. The affinity of RBD for effector region mutants or the GDP-bound form of p21ras is in the micromolar range, which means that 100-fold lower affinity is not sufficient for signal transduction. The rate of the GTPase of p21ras is not modified by RBD. Since P(i) release is found not to be rate limiting, the Ras-Raf signal of the cell may be terminated by the intrinsic GTPase of p21ras.
Herrmann
C
C
Max-Planck-Institut für Molekulare Physiologie, Dortmund, Federal Republic of Germany.
Martin
G A
GA
Wittinghofer
A
A
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020558:GTP Phosphohydrolases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)