Figure 5.

Activation of PKCη by β1γ2, and subsequent hyper phosphorylation of PKD in the activation loop. HeLa cells were cotransfected with the constructs listed. The cells were lysed and the extracts analyzed by Western blotting to monitor the phosphorylation status of FLAG-PKCη, GFP-PKCɛ, and GST-PKD, respectively. The blots were quantitated as described in Materials and methods. (A) For PKCη, the antibody used recognizes threonine 655 (T655). (B) Similar experimental procedure was used to monitor the effect of β1γ2 expression on the phosphorylation status of PKCɛ phosphorylation (Ser729). (C) Coexpression of β1γ2 and PKCη caused a fourfold increase in the phosphorylation of Ser744/748 (in the activation loop) of PKD without any appreciable change in the autophosphorylation of Ser916 (lane 2, shown in the Western blot and the bar graph). Values are means (±SD, vertical bars) of three separate experiments.