PKCζ interacts with ERK5. (A) ERK5 binds to PKCζ in vitro. HeLa cells were cotransfected with HA-PKCζ and pcDNA3 or Flag-ERK5 for 24 hours and subjected to immunoprecipitation with Flag antibody, followed by Western blot analysis with HA antibody. Expression of ERK5 and PKCζ was detected by Western blotting with specific antibodies. (B) TNFα slightly increases PKCζ-ERK5 binding. Subconfluent cocultures of HUVECs were treated for different time points with TNFα 10 ng/mL. The interaction of endogenous PKCζ with endogenous ERK5 was evaluated by immunoprecipitating 400 μg of total cell lysate with ERK5 antibody, and the immunoprecipitates were analyzed by immunoblotting with PKCζ antibody. Essentially identical results were obtained in 2 other experiments. (C,E) The COOH-terminus regions of PKCζ and ERK5 are critical for the ERK5-PKCζ interaction. HUVECs were transfected with plasmids expressing wild-type VP16-ERK5 with Gal4-PKCζ fragments (C) or wild-type Gal4-PKCζ with VP16-ERK5 fragments (E) as indicated, and luciferase activity was evaluated 24 hours after transfection. (D) PKCζ fragment able to bind ERK5 inhibits ERK5 transactivation. HUVECs were transfected with pBIND-ERK5 and Gal4-dependent (pG5-Luc) reporter gene with or without pcDNA3-CA-MEK5α with PKCζ-3 and PKCζ-2 fragments. Luciferase activity was measured after 24 hours of incubation. Data are mean ± SD of 3 experiments performed in triplicate. **P < .05.