Induction of nonapoptotic cell death by activated Ras requires inverse regulation of Rac1 and Arf6.
Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin-independent 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.
Bhanot
Haymanti
H
Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, Ohio 43614, USA.
Young
Ashley M
AM
Overmeyer
Jean H
JH
Maltese
William A
WA
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors* / Q000502:physiology; D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology; D017209:Apoptosis / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000502:physiology; D016923:Cell Death / Q000235:genetics; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000235:genetics*; D004705:Endocytosis / Q000235:genetics; D011992:Endosomes / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D004789:Enzyme Activation / Q000235:genetics; D005909:Glioblastoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D057809:HEK293 Cells; D006801:Humans; D012516:Osteosarcoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D014617:Vacuoles / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism* / Q000502:physiology; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D000089982:ADP-Ribosylation Factor 6; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C485647:GIT1 protein, human; C107953:RAC1 protein, human; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020830:rac1 GTP-Binding Protein; D018631:ras Proteins