Induction of Nonapoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

Abstract

Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrinindependent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1 (G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.

Unravelling the Mechanism of TrkA-Induced Cell Death by Macropinocytosis in Medulloblastoma Daoy Cells

“…Under normal conditions where the macropinosome matures and is then recycled back up to the cell membrane, activation of Arf6 is again required for this final step and membrane fusion (54,78). In U251 cells, the resting activity of Arf6 is high but drops significantly upon stimulation of macropinocytosis (23). In contrast to these results, we observed no change in the activation levels of Arf6 in NGF-stimulated Daoy-TrkA cells.…”

“…The ability of DNRab5 to block NGF-induced macropinosomes is consistent with its known roles in mediating the early fusion of endosomes (15) and H-Ras-induced macropinosomes (49). With respect to DNRac1, as stated earlier, CA-Ras-dependent activation of macropinocytosis in U251 cells increased the pool of active Rac1 (21,23), which is known to serve a role in membrane ruffling and the formation of lamellipodia (50,51,52). In contrast, we found that the levels of NGF-activated Rac1 in Daoy-TrkA cells were relatively low by 12 to 24 h (Fig.…”

“…1). NGFdependent cell death in Daoy-TrkA cells was strikingly similar to that observed in the GB cell line U251 following expression of activated H-Ras (20,21,23). Similar to findings in U251 cells, macropinocytosis in Daoy-TrkA cells is also dependent on the activation of H-Ras.…”

“…H-Ras has been shown to have a diversity of roles in different cell types, including inducing macropinocytosis to facilitate nutrient uptake in pancreatic tumors (14), in addition to its well-known roles in regulating cell survival (70), senescence (71), and cell death (72). As described earlier, H-Ras has been shown to stimulate macropinocytosis in U251 cells via activation of Rac1 (23), and expression of CA-Rac1 can itself stimulate macropinocytosis and cell death (21). This is in contrast to our observations that expression of CA-Rac1 had no effect on macropinosome formation and that the expression of DN-Rac1 and/or depletion of endogenous Rac1 by siRNA did not affect NGF-induced macropinocytosis.…”

Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT–PIX complexes

“…In GIT-PIXdependent systems, elevated Rac1 and/or Cdc42 activity has been found to be associated with reduced Arf6 activity (Albertinazzi et al, 2003;Hajdo-Milasinovic et al, 2009;Jones et al, 2009;Bhanot et al, 2010). However, a coordinated regulation, as opposed to merely a colocalization of GAP and GEF functions, has not been demonstrated.…”