Grb4 expression does not activate the JNK, Elk1, or the CREB signaling pathway but inhibits v-Abl–induced AP-1 activation.
(A-C) JNK, Elk1, and CREB pathway. We transiently transfected 1 × 105 293 cells with a plasmid expressing Grb4, a beta-galactosidase construct to normalize for transfection efficiency, and a construct for the expression of a fusion transcription factor encoding the DNA-activation domain of Jun, Elk, or CREB and the DNA-binding domain of GAL4. The luciferase gene was under the control of the GAL4 promoter. As a positive control, mitogen-activated protein kinases (MEKK in panel A, MEK1 in panel C) and protein kinase A (PKA in panel B) were used. Results are representative of at least 3 independent experiments. (D) AP-1 activation. We transiently transfected 293 cells with 1 μg of a plasmid expressing v-abl or kinase-inactive v-abl together with increasing amounts of a plasmid expressing Grb4 as indicated, beta-galactosidase, and a reporter construct with 7 AP-1 sites upstream of the luciferase gene. In each reaction, equal amounts of DNA were transfected. Results are the mean and SD determined from an experiment done in triplicate reproduced at least 3 times.
