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Proteolytic cleavage of ras GTPase-activating protein during apoptosis
L P Wen1, K Madani, G A Martin
1Department of Pulmonary and Critical Care Medicine, Stanford University, Stanford, California 94305-5236, USA.
Abstract:
p120-ras GTPase-activating protein (rasGAP) associates with Ras and negatively regulates Ras signaling by stimulating the intrinsic rate of Ras GTPase activity. rasGAP also associates with other cellular signaling proteins which suggest that rasGAP may play a role in coordinating other signal transduction pathways. Disruption of rasGAP in vivo results in extensive apoptosis. Fas-mediated apoptosis results in the activation of caspases that cleave cellular substrates which are important for maintaining cytoplasmic and nuclear integrity. We show here that rasGAP is proteolytically cleaved by caspases early in Fas-induced apoptosis of Jurkat cells. rasGAP was also cleaved by DNA-damaging chemotherapeutic agents and TNF-related apoptosis inducing ligand (TRAIL), also known as Apo2L. Based on the size of the products generated by cleavage of deletion mutants of rasGAP we predict that cleavage of rasGAP occurs in the hydrophobic region and between the SH2(2) and ras-p21 interacting domain which would leave an intact ras-p21 interacting domain. Interestingly, cleavage of rasGAP in vitro enhanced rasGAP hydrolysis activity. Our results demonstrate that diverse apoptotic stimuli cause caspase-mediated cleavage of rasGAP early in apoptosis.
Insights
Diverse apoptotic signals cleave p120-ras GTPase-activating protein (rasGAP) via caspases early in apoptosis. This cleavage enhances rasGAP
Area of Science:
- Cellular signaling
- Apoptosis
- Molecular biology
Background:
- p120-ras GTPase-activating protein (rasGAP) negatively regulates Ras signaling by stimulating Ras GTPase activity.
- rasGAP interacts with other signaling proteins, suggesting a role in coordinating signal transduction pathways.
- Disruption of rasGAP in vivo leads to apoptosis.
Purpose of the Study:
- To investigate the role of rasGAP cleavage during apoptosis.
- To determine if caspases cleave rasGAP during Fas-induced apoptosis.
- To identify the cleavage sites and functional consequences of rasGAP cleavage.
Main Methods:
- Apoptosis induction in Jurkat cells using Fas-mediated pathways, chemotherapeutic agents, and TNF-related apoptosis-inducing ligand (TRAIL).
- Analysis of rasGAP cleavage products using deletion mutants.
- In vitro assays to assess the effect of cleavage on rasGAP hydrolysis activity.
Main Results:
- rasGAP is proteolytically cleaved by caspases early in Fas-induced apoptosis.
- Cleavage also occurs upon treatment with DNA-damaging agents and TRAIL.
- Predicted cleavage sites are in the hydrophobic region and between SH2(2) and the ras-p21 interacting domain.
- Cleavage enhances rasGAP hydrolysis activity in vitro.
Conclusions:
- Diverse apoptotic stimuli trigger caspase-mediated cleavage of rasGAP.
- Cleavage occurs early in the apoptotic process.
- The cleavage of rasGAP may modulate its function and contribute to apoptotic signaling.