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Comparing the Affinity of GTPase-binding Proteins using Competition Assays
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Regulation of local GTP availability controls RAC1 activity and cell invasion.
Anna Bianchi-Smiraglia1, David W Wolff2, Daniel J Marston3
1Department of Cell Stress Biology, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA. Anna.Bianchi-Smiraglia@RoswellPark.org.
Nature Communications
|October 20, 2021
Summary
Cellular GTP levels regulate RAC1 activation by influencing local GTP availability near the RAC1-GTP dissociation constant. This study reveals a novel mechanism involving guanylate metabolism enzymes like IMPDH2 in controlling RAC1 activity and cell invasion.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Intracellular GTP concentration was previously thought too high to regulate RAC1 activation.
- RAC1 activation is crucial for various cellular processes, including cell motility and invasion.
Purpose of the Study:
- To investigate the role of physiological GTP level changes in regulating RAC1 activation in live cells.
- To elucidate the mechanism by which local GTP pools control RAC1 activity.
Main Methods:
- Utilized genetically encoded GTP biosensors and a RAC1 activity biosensor in live-cell imaging.
- Investigated the interaction between RAC1 and guanylate metabolism enzymes, specifically IMPDH2.
- Employed mutant IMPDH2 and targeted IMPDH2 localization to assess its role in GTP regulation and RAC1 activity.
Main Results:
- Demonstrated correlation between fluctuating GTP levels around the RAC1-GTP Kd and RAC1 activity.
- Identified co-localization of RAC1 with IMPDH2 in cell protrusions, mediated by direct interaction.
- Showed that IMPDH2 binding to RAC1 is essential for RAC1 activity, independent of total cellular GTP levels.
- Found that mislocalization of IMPDH2 reduced local GTP levels, RAC1 activity, and cell invasion.
Conclusions:
- Local GTP pools, not just total cellular concentration, regulate RAC1 activity.
- IMPDH2 plays a critical role in modulating local GTP availability and thereby controlling RAC1-mediated cellular functions like invasion.
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