Direct measurement of the binding of RAS to neurofibromin using a scintillation proximity assay.
Protein-protein interactions are of major importance in many cellular processes. When no enzymic activity is involved, assays for direct binding are required. One such example is the relatively weak interaction between oncogenic Ras and the GTPase-activating protein neurofibromin (NF1). The complex between the catalytic domain of NF1 and the GTP-form of oncogenic Ras protein dissociates rapidly; hence, equilibrium binding must be quantitated. Scintillation proximity assay (SPA) technology, a radioisotopic technique that requires no separation step, was used to characterize this interaction. Leu-61 Ras complexed with [3H]GTP was generated by nucleotide exchange in the presence of a GTP-regenerating system. A SPA signal was obtained when radiolabeled Ras was mixed with NF1 fused with glutathione S-transferase (GST), anti-GST, and protein A-coated SPA beads. This signal was abolished when any of the components were omitted and also by the addition of NaCl, which potently reduces the affinity of interaction between Ras and NF1. The neutralizing anti-Ras monoclonal antibody Y13-259 and the detergent n-dodecyl maltoside, a specific inhibitor of NF1 catalytic activity, both abolished the SPA signal from the NF1/Ras assay but neither affected a control SPA signal in which a [3H]GTP.GST-Ras fusion protein was bound to protein A-coated SPA beads. This technology could be readily extended to the measurement of other protein-protein interactions and could form the basis for high-throughput screens for the discovery of novel therapeutic agents.
Skinner
R H
RH
Department of Cell Biology, Wellcome Research Laboratories, Beckenham, Kent, United Kingdom.
Picardo
M
M
Gane
N M
NM
Cook
N D
ND
Morgan
L
L
Rowedder
J
J
Lowe
P N
PN
D004926:Escherichia coli / Q000235:genetics; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D025542:Neurofibromin 1; D011485:Protein Binding; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012588:Scintillation Counting / Q000379:methods*; D014316:Tritium; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D025542:Neurofibromin 1; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D014316:Tritium; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D018631:ras Proteins