Gorman C, Skinner R H, Skelly J V, Neidle S, Lowe P N
Wellcome Research Laboratories, Langley Court, South Eden Park Road, Beckenham, Kent BR3 3BS, United Kingdom.
J Biol Chem. 1996 Mar 22;271(12):6713-9. doi: 10.1074/jbc.271.12.6713.
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.
Raf是一种丝氨酸/苏氨酸激酶，它通过其氨基末端调节结构域与GTP形式的Ras结合，从而激活丝裂原活化蛋白激酶途径。在本研究中，我们使用平衡结合方法（闪烁邻近分析和荧光偏振）而非更广泛使用的非平衡方法（如酶联免疫吸附测定和亲和沉淀）来表征Raf的Ras结合结构域与Ras的相互作用。最初使用与Raf的1-257或1-190残基的谷胱甘肽S-转移酶融合蛋白进行的研究表明，虽然使用酶联免疫吸附测定或亲和沉淀可以检测到Ras结合，但这是亚化学计量的；在仅少量过量Raf的平衡条件下，几乎检测不到结合。这种差异可能是由于存在高比例的无活性Raf蛋白。进一步的研究使用了含有Raf的51-131残基的蛋白质，该蛋白质在大肠杆菌中作为稳定的谷胱甘肽S-转移酶融合蛋白表达。使用这种蛋白质，可以在平衡条件下轻松测量与Ras的结合。神经纤维瘤蛋白的催化结构域抑制Ras与Raf的结合，而Raf抑制Ras与神经纤维瘤蛋白的结合，这表明Raf和神经纤维瘤蛋白不能同时与Ras结合。神经纤维瘤蛋白和Raf与Harvey-RasLeu-61的相互作用亲和力相似。Raf从Ras解离的速率常数估计大于1分钟-1，这表明Ras、Raf和神经纤维瘤蛋白在细胞中可能处于快速平衡状态。与之前的报道相反，在平衡条件下，没有证据表明Raf的最小Ras结合结构域（51-131残基）与包含额外16个羧基末端氨基酸的区域之间的亲和力存在差异，这表明132-147残基不构成关键的结合决定因素。
