Impaired Akt activity down-modulation, caspase-3 activation, and apoptosis in cells expressing a caspase-resistant mutant of RasGAP at position 157.
RasGAP bears two caspase-3 cleavage sites that are used sequentially as caspase activity increases in cells. When caspase-3 is mildly activated, RasGAP is first cleaved at position 455. This leads to the production of an N-terminal fragment, called fragment N, that activates the Ras-PI3K-Akt pathway and that promotes cell survival. At higher caspase activity, RasGAP is further cleaved at position 157 generating two small N-terminal fragments named N1 and N2. We have now determined the contribution of this second cleavage event in the regulation of apoptosis using cells in which the wild-type RasGAP gene has been replaced by a cDNA encoding a RasGAP mutant that cannot be cleaved at position 157. Our results show that cleavage of fragment N at position 157 leads to a marked reduction in Akt activity. This is accompanied by efficient processing of caspase-3 that favors cell death in response to various apoptotic stimuli. In nontumorigenic cells, fragments N1 and N2 do not modulate apoptosis. Therefore, the role of the second caspase-mediated cleavage of RasGAP is to allow the inactivation of the antiapoptotic function of fragment N so that caspases are no longer hampered in their ability to kill cells.
Yang
Jiang-Yan
JY
Department of Cellular Biology and Morphology, Biology and Medicine Faculty, Lausanne University, 1005 Lausanne, Switzerland.
Walicki
Joël
J
Michod
David
D
Dubuis
Gilles
G
Widmann
Christian
C
D000818:Animals; D017209:Apoptosis; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D015536:Down-Regulation; D004789:Enzyme Activation; D005347:Fibroblasts; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics*; D020703:ras GTPase-Activating Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*
D020703:ras GTPase-Activating Proteins; D001224:Aspartic Acid; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases