Mechanism of ERM protein–dependent CD44 cleavage. Mechanism of ERM protein–dependent CD44 cleavage. A, inhibition of the Ras pathway does not affect CD44 cleavage. NIH3T3 cells were seeded in 6-well plates at low cell density. The cells were transfected as in Fig. 1A. Cells were pretreated with increasing concentrations of MEK1 inhibitor (PD98059) for 30 minutes and afterward stimulated with 100 ng/mL TPA for 4 hours. The effect of PD98059 was confirmed by its inhibition of Erk phosphorylation. Inhibition of the MEK-ERK pathway had no effect on CD44 cleavage. B, effect of overexpressed ezrin mutants on CD44 cleavage. RPM-MC cells were cotransfected with plasmids encoding N-terminal FLAG and C-terminal HA-tagged CD44 and plasmids encoding myc-tagged ezrin mutants (described in ref. 46): T567A (inactive), T567D (active), R579A (not interacting with F-actin) and treated as in Fig. 1A. Overexpressed mutant ezrins compete with endogenous ERM proteins for cleavage-relevant interactions. C, block of actin polymerization inhibits CD44 cleavage. RPM-MC cells were transfected with a plasmid encoding wild-type CD44 and treated with DAPT as in Fig. 1A. To block actin polymerization (F-actin), latrunculin B was added at the concentrations indicated. Thirty minutes after addition of latrunculin B, cells were stimulated with 100 ng/mL TPA for 30 minutes. D, neuregulin release is inhibited by interference with F-actin. HEKNE wt cells (HEK293T-AT1R +pB-Flag-NRG1-EGFP) were pretreated with a vehicle control (DMSO) or with the Arp2/3 inhibitor CK-548 in increasing concentrations (0–15 μmol/L) alone or before angiotensin II (1 nmol/L) stimulation. NRG1 cleavage was detected by immunoblotting using NRG1 antibodies. Angiotensin II was able to induce NRG1 cleavage in the absence (lane 2) but not in the presence of CK-548 (lanes 6–8). Monika Hartmann et al. Mol Cancer Res 2015;13:879-890 ©2015 by American Association for Cancer Research