FIGURE 10.

PAR₂-mediated ERK2 activation is not regulated by AMSH or UBPY catalytic activities. A, HEK-PAR₂ cells were incubated with vehicle (DMSO; 1:10,000) or 1 μm AG1478 for 1 h prior to stimulation with AP (100 μm) or EGF (100 ng/ml) for 5 min. Cell lysates were analyzed by Western blotting for pERK and ERK2. Densitometry indicated that AG1478 abolished EGF-dependent ERK2 activation but had little effect on AP-dependent ERK2 activation. n = 3. HEK cells were transiently transfected with PAR₂ and pcDNA5 (control), GFP-AMSH(D348A), or GFP-UBPY(C786S). Serum-starved cells were challenged with AP (100 μm, 0–10 min), washed, and incubated in AP-free medium (0–180 min). pERK and ERK2 levels were analyzed by Western blotting (B) and quantified using densitometry (C). The levels of ERK2 activation in cells expressing GFP-AMSH(D348A) or GFP-UBPY(C786S) were similar to control cells. n = 3.