Figure 6.

Regulation of ERK6 (p38γ) by molecules acting downstream from Rho. (A) NIH-3T3 (top) and HEK-293T cells (bottom) were transfected with expression vectors containing HA-tagged ERK6 along with RhoA QL, PKN wt, PKN Δ ROKα wt, ROKα Δ, or MEK3 EE (1 μg each), as indicated. Kinase and Western blotting assays were performed as described above. Histograms represent the kinase activity relative to that in cells transfected with empty expression vector (control), whose value was taken as 1. Gels are representative of three independent experiments. (B) Lysates from 293T cells transfected with the indicated expression vectors were analyzed by Western blotting by use of anti-PKN and anti-tag (myc) antibodies. (C) NIH-3T3 cells were transfected as above with pSREmutL (0.1 μg per plate) or pJLuc together with pRL-null reporter plasmid DNAs (0.01 μg per plate) and treated with 10 μM LPA for 5 h or cotransfected along with PKN Δ alone or in combination with MKK3 AA (1 μg), as indicated. Transfection with MKK3 EE served as a control. Twenty-four hours after transfection, cells were collected and the lysates were assayed for dual luciferase activities. The data represent firefly luciferase activity normalized by Renilla luciferase activity present in each sample expressed as fold induction relative to control, and are the average ± s.e. of triplicate samples from a typical experiment. Similar results were obtained in three independent experiments.