Figure 3. E1A acts upstream of JNK through a Rac1-MEKK1-MKK4/7 pathway.

(A) E1A activates MKK4 and MKK7. The activities of immunoprecipitated HA-MKK4 and myc-MKK7 were determined by an in vitro protein kinase assay with GST-JNK1 as a substrate. (B) c-Jun-GAL4 reporter assay of cells co-transfected with the plasmids encoding for E1A-ER and dominant-negative mutants (DN mut) of JNK upstream signaling components. (C) Histogram showing the c-Jun-GAL4 reporter assay after depletion of RhoA, Rac1 or Cdc42 by siRNA. Right panels: immunoblots of the depleted proteins. (D) Pull-down of GTP-loaded Rac1 with GST-human Pak1-PBD as a substrate. Lower panels: control immunoblots of co-transfected E1A-ER and myc-Rac1. In all histograms: one-tailed p-values were calculated by Student's t-test; error bars represent s.d. Cells were incubated with E₂ for 7 h. NT, NonTarget siRNA.