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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates
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Tumor-derived RHOA mutants interact with effectors in the GDP-bound state
Yuan Lin1, Theresa A Ramelot2, Simge Senyuz3
1Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. yuan.lin@cchmc.org.
Nature Communications
|August 21, 2024
Summary
Gain-of-function mutations in RHOA (Ras homolog family member A) accelerate its activity, enabling effector interaction even in the GDP-bound state, contributing to cancer development.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Ras homolog family member A (RHOA) mutations are implicated in various cancers.
- Specific RHOA mutations, A161P and A161V, are found in adult T-cell leukemia/lymphoma.
Purpose of the Study:
- To investigate the molecular mechanisms of two gain-of-function RHOA mutations, A161P and A161V.
- To understand how these mutations contribute to tumorigenesis in adult T-cell leukemia/lymphoma.
Main Methods:
- Biochemical assays to measure guanine nucleotide exchange and GTP hydrolysis rates.
- X-ray crystallography to determine the structures of RHOA mutants.
- Nuclear Magnetic Resonance (NMR) spectroscopy (31P and 1H-15N HSQC) to study protein dynamics.
- Molecular dynamics simulations to analyze conformational changes.
Main Results:
- RHOA(A161P) and RHOA(A161V) exhibit fast cycling rates and reduced GTPase activity compared to wild-type RHOA.
- Crystal structures reveal altered nucleotide binding in RHOA(A161P) and an open nucleotide pocket in RHOA(A161V).
- Mutations destabilize RHOA switch regions, favoring an active conformation and enabling effector interaction in the GDP-bound state.
Conclusions:
- The A161P and A161V mutations confer gain-of-function properties to RHOA by altering its nucleotide binding and dynamics.
- These mutations promote constitutive RHOA activity, potentially driving tumorigenesis through aberrant effector interactions.
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