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. 2018 May 11;293(26):10172–10185. doi: 10.1074/jbc.RA117.000327

Figure 3.

Figure 3.

RHOA is a downstream target of DAAM1, and its activation is required for collagen-induced cell haptotaxis. A–C, RHOA, RAC1, and CDC42 activations were elevated by type IV collagen treatment. MDA-MB-231 cells were seeded on Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA, RAC1, and CDC42 activation assays. D, collagen-induced RHOA activation was inhibited by DAAM1 silence. Stable DAAM1 knockdown MDA-MB-231 cells (DAAM1 shRNA #1 and #2) and control cells were seeded on Boyden chamber membranes coated with collagen type IV on the lower sides. Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA, RAC1, and CDC42 activation assays. E, collagen-induced RHOA activation was inhibited by cyclo(-RGDfK) treatment. MDA-MB-231 cells were seeded on Boyden chamber membranes coated with collagen type IV on the lower sides. Cells treated with cyclo(-RGDfK) (20 nmol/liter) were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA activation assays. Cy, cyclo(-RGDfK). F and G, CCG-1423 (RHOA-specific inhibitor) or RHOA-N19 (dominant-negative RHOA) inhibited collagen-induced haptotaxis of MDA-MB-231 cells. MDA-MB-231 cells treated with CCG-1423 (1 μmol/liter) or RHOA-N19–overexpressing stable cells were seeded in Boyden chamber membranes coated with type IV collagen on both sides (Both) or lower sides (Lower). Cells were allowed to migrate toward collagen for 8 h. H, CCG-1423 or RHOA-N19 inhibited collagen-induced haptotaxis of MDA-MB-453 cells. MDA-MB-453 cells treated with CCG-1423 (1 μmol/liter) or transfected with RHOA-N19 were seeded in Boyden chamber membranes coated with type IV collagen on both sides (Both) or lower sides (Lower). Cells were allowed to migrate toward collagen for 8 h. I and J, DAAM1 activation was not altered by RHOA-N19 overexpression. RHOA-N19–overexpressing stable cells were seeded on Boyden chamber membranes coated with type IV collagen on both sides (Both) or on lower sides (Lower). Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. K, RHOA-V14 rescued the haptotaxis of N-DAAM1–expressed MDA-MB-231 cells. MDA-MB-231 cells transfected with N-DAAM1 and/or RHOA-V14 were seeded in Boyden chamber membranes coated with type IV collagen on lower sides. Cells were allowed to migrate toward collagen for 8 h. Error bars, S.D.