Figure 5.

In vitro activation of SOS2‐∆308 by GRIK1.

The GST‐fused SOS3‐independent form of SOS2 (SOS2‐∆308, arrowhead), or the combined T168A mutation in SOS2‐∆308 were incubated with GST‐fused GRIK1 or GST‐fused kinase‐dead (K137R) version of GRIK1 (GRIK1‐KR). The SOS1 C‐terminal fragment (SOS1‐CT, arrows) was added as a phosphorylation substrate of SOS2. Proteins were separated in sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by Coomassie staining (left) and autoradiograph (right). Note that the small amount of GRIK1 used was enough to achieve the activation of SOS2, even though GRIK1 bands were not observed. The relative band intensity of SOS1‐CT bands was normalized to the signal from the SOS2‐∆308WT/GRIK1‐KR lane (mean ± SE, n = 6). Asterisks indicate significant differences (P < 0.05, binomial test).