Dsg3-induced Ezrin-Thr567 phosphorylation could be abrogated by various inhibitors including those for PKCs. (
a) Western blotting of total lysates of A431 lines treated with the broad spectrum PKC inhibitor, bisindolylmakeimide I (BIM), at various concentrations (0.5, 5, 10, 20 and 40 μ M) for 4 h that showed a dose-dependent inhibition of pERM levels in all cell lines. Note that higher concentrations (<20 μ M) of the inhibitor were required to abrogate the enhanced pERM in Dsg3-overexpressing cells (D3 and C7) compared with Vect and C11 cells that were treated at lower concentrations (<10 u M). In addition, all Dsg3-transducted cells appeared to be more sensitive than control cells to BIM. The results are representative of at least three independent experiments. ( b) Quantitation of the western blots of a time course experiment in which four cell lines were stimulated in normal growth medium for 3 h after serum starvation, before treatment with BIM for 0.5 and 1 h. Again, less inhibition was seen in Dsg3-overexpressing cell lines. ( c) The phosphorylation of the ERM proteins can be abrogated by the inhibitors for various signaling molecules in addition to PKCs. Cells were serum starved for 1 day before being treated with the inhibitors as follows: PKC-Rö 31-7549: 2 μ M for 5 h or 50 μ M for 45 min, PCK-BIM: 5 μ M for 4 h, Src-PP2: 10 μ M for 5 h, RhoA-C3: 2 μg for 5 h, ROCK-Y-27652: 10 μ M for 4 h, Rac1-NSC23766: 30 μ M for 5 h and p38-SB202190: 20 μ M for 4 h. Lysates were extracted and subsequently analyzed by western blotting. Except for Src inhibitor PP2, almost all the inhibitors showed inhibition of pERM to different degrees. The results are representative of three independent experiments, and shown below are the quantitation data (mean±s.d.).