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RhoC GTPase Activation Assay
Published on: August 23, 2010
The function of the p190 Rho GTPase-activating protein is controlled by its N-terminal GTP binding domain
N Tatsis1, D A Lannigan, I G Macara
1Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, USA.
The Journal of Biological Chemistry
|December 16, 1998
Summary
The N-terminal GTP binding domain of p190 regulates its Rho/Rac GTPase-activating protein (GAP) activity within cells. This regulation is crucial for cell morphology and Jun kinase (JNK) activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- p190 functions as a GTPase-activating protein (GAP) for Rho family GTPases.
- The N-terminal GTP binding domain of p190's function was previously unknown.
Purpose of the Study:
- To investigate the role of the N-terminal GTP binding domain of p190.
- To determine how this domain affects p190's Rho/Rac GAP activity in cellular contexts.
Main Methods:
- Site-directed mutagenesis (Ser36 to Asn) in the N-terminal GTP binding domain.
- Expression of wild-type and mutant hemagglutinin (HA)-tagged p190 in COS and NIH 3T3 cells.
- Assessment of GAP activity, cell morphology, and Jun kinase (JNK) activation.
Main Results:
- A mutation (S36N) in the N-terminal domain reduced guanine nucleotide binding and impaired p190's cellular RhoGAP function.
- Wild-type p190 induced cell rounding and beaded extensions, phenotypes dependent on GAP activity and suppressible by Rho/Rac activation.
- The N-terminal domain fragment partially inhibited the phenotype, suggesting it sequesters a required factor.
Conclusions:
- The N-terminal GTP binding domain of p190 regulates its Rho/Rac GAP activity in vivo.
- This regulation impacts cell morphology and JNK signaling pathways.
- p190's N-terminal domain may sequester regulatory factors essential for its function.
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