Abstract
Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway. In this study, we have characterized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluorescence anisotropy), rather than with more widely used nonequilibrium procedures (such as enzyme-linked immunosorbent assay and affinity precipitation). Initial studies using glutathione S-transferase fusion proteins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosorbent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no binding was detected. This difference was probably due to the presence of a high percentage of inactive Raf protein. Further studies used protein containing residues 51-131 of Raf, which expressed in Escherichia coli as a stable glutathione S-transferase fusion. With this protein, binding with Ras could readily be measured under equilibrium conditions. The catalytic domain of neurofibromin inhibited binding of Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin showing that Raf and neurofibromin cannot be bound simultaneously to Ras. The affinities of interaction of neurofibromin and Raf with Harvey-RasLeu-61 were similar. The rate constant for dissociation of Raf from Ras was estimated to be >1 min-1, suggesting that Ras, Raf, and neurofibromin may be in rapid equilibrium in the cell. In contrast to previous reports, under equilibrium conditions there was no evidence for a difference in affinity between the minimal Ras binding domain of Raf (residues 51-131) and a region containing an additional 16 carboxyl-terminal amino acids, suggesting that residues 132-147 do not form a critical binding determinant.
Highlights
The proto-oncogene ras encodes a membrane-associated guanine nucleotide binding protein (Mr 21,000) that is activated by mutation in approximately 30% of human tumors
The GST-raf-1 proto-oncogene encodes a serine/threonine kinase (Raf)(1–257) was about 90% pure (Fig. 1, track 2), whereas the GST-Raf-(1–190) eluted as a broad peak and the preparation still contained many contaminating proteins (Fig. 1, track 4). The ability of these Raf fusion proteins to bind to Ras was initially monitored using ELISA, similar to that used by Ghosh et al (25)
Using ELISA and affinity precipitation techniques, these longer fragments appear to be unable to bind Ras stoichiometrically. Both techniques involve separation steps and do not measure equilibrium binding but the amount of binding detected in a scintillation proximity assay under equilibrium conditions was much lower than expected for the amount of Raf proteins added
Summary
The proto-oncogene ras encodes a membrane-associated guanine nucleotide binding protein (Mr 21,000) that is activated by mutation in approximately 30% of human tumors (for reviews, see Refs. 1– 4). It acts as a molecular switch in signaling from growth factor receptors at the cell membrane to nuclear transcription factors via the cytoplasmic mitogen-activated protein kinase cascade (5, 6). Activation of a growth factor receptor by ligand binding results in the conversion of Ras from its inactive GDP-bound to active GTP-bound state. This occurs through nucleotide exchange catalyzed by exchange factors. Much of the previously reported data comparing the interactions of different Raf constructs with Ras have been obtained using nonequilibrium binding procedures involving separation steps (13, 23, 25–28), and little attempt has been made to ensure that all Raf proteins are active on a molar basis with respect to Ras binding.
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