FIG. 4.

A C282R mutation in hSOS1 is functionally equivalent to the sos-1(sy262) G322R mutation in C. elegans SOS-1. (A) hSOS1 C282R promotes EGF-dependent MAPK activation in NIH 3T3 cells. The cells were transfected with 2 to 4 μg of FLAG-tagged hSOS1 expression vector and 4 μg of HA-ERK1 (MAPK) expression vector. After serum starvation and stimulation with EGF for the indicated times, whole-cell lysates were prepared and analyzed by Western blotting with the indicated antibodies. Phosphorylated and total transfected MAPKs were distinguished from endogenous MAPK by a mobility shift caused by the HA tag on the MAPK in the transfection construct. Representative blots from two independent experiments are shown. Activation (n-fold) was calculated by dividing the α-phospho-MAPK signal by the α-total-MAPK signal and then adjusting the values to that observed with wild-type hSOS1 at the zero time point. (B) Mean results from four independent experiments in NIH 3T3 cells, where the wild-type and mutant hSOS1 constructs were similarly expressed and the control wild-type hSOS1 construct caused similar levels of MAPK activation between experiments. Normalized activation (n-fold) is as described for panel A, except that α-phospho-MAPK/α-total MAPK ratios were also adjusted for slight differences in transfected hSOS1 expression. Statistical significance was assessed using a one-tailed Student's t test. (C) hSOS1 C282R promotes EGF-dependent MAPK activation in HEK 293 EBNA cells. The cells were transfected with 2 to 4 μg of FLAG-tagged hSOS1 expression vector and 2.5 μg of HA-ERK1 (MAPK) expression vector. After serum starvation and stimulation with EGF for the indicated times, whole-cell lysates were prepared and analyzed by Western blotting as described for panel A. Representative blots from two independent experiments are shown.