EQUILIBRIUM AND KINETIC MEASUREMENTS REVEAL RAPIDLY REVERSIBLE BINDING OF RAS TO RAF

Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mi togen activated protein kinase pathway, In this study, we have charact erized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluore scence anisotropy), rather than with more widely used nonequilibrium p rocedures (such as enzyme-linked immunosorbent assay and affinity prec ipitation). Initial studies using glutathione S-transferase fusion pro teins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosor bent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no bindi ng 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 col i as a stable glutathione S-transferase fusion, With this protein, bin ding 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 Ra f and neurofibromin cannot be bound simultaneously to Ras, The affinit ies of interaction of neurofibromin and Raf with Barvey-Ras(Leu-61) we re similar, The rate constant for dissociation of Raf from Ras was est imated to be >1 min-l, 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 i n 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.