Figure 4.

SIMKK enhances the activation of SIMK and SAMK by CuCl₂. SIMK-HA (A) and SAMK-HA (B) were either expressed alone (1, 5, and 9) or coexpressed with myc-tagged MEK1 (2, 6, and 10), PRKK (3, 7, and 11), or SIMKK (4, 8, and 12) in protoplasts. The protoplasts were treated either for 30 min with 100 μm CuCl₂ (lanes 5–8), 100 μm CdCl₂ (lanes 9–12), or as control with equal amount of water (lanes 1–4). Subsequently, SIMK and SAMK activities were determined by immunokinase assays using anti-HA antibodies for immunoprecipitation and MBP as substrate. The same protein extracts were analyzed for the presence of the coexpressed proteins by protein gel-blot analysis with anti-HA or anti-Myc antibodies. Three independent experiments yielded comparable results.