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Identification of Aurora kinases as RasGAP Src homology 3 domain-binding proteins
Veronique Gigoux1, Sebastien L'Hoste, Francoise Raynaud
1Laboratoire de Pharmacochimie Moléculaire et Structurale, CNRS FRE2463-INSERM U266, UFR des Sciences Pharmaceutiques et Biologiques, 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France.
Abstract:
The GTPase-activating protein RasGAP functions as both a negative regulator and an effector of Ras proteins. In tumor cells, RasGAP is no longer able to deactivate oncogenic Ras proteins, and its effector function becomes predominant. As RasGAP itself has no obvious enzymatic function that may explain this effector function, we looked for downstream RasGAP effectors that could fulfill this role. We looked for the existence of RasGAP Src homology 3 (SH3) domain partners as this domain is involved in the regulation of cell proliferation and has an anti-apoptotic effect. We report here the identification of a new RasGAP SH3 domain-binding protein, named Aurora. This Drosophila melanogaster Ser/Thr kinase has three human orthologs called Aurora/Ipl1-related kinase or HsAIRK-1, -2, and -3. Coimmunoprecipitation experiments in COS cells confirmed that HsAIRK-1 and HsAIRK-2 both interact with RasGAP. RasGAP pull-down experiments showed that it interacts with HsAIRK-1 in G(2)/M HeLa cells. We also demonstrated that RasGAP binds to the kinase domain of Aurora and that this interaction inhibits the kinase activity of HsAIRK-1 and HsAIRK-2. Finally we showed that RasGAP forms a ternary complex with HsAIRK and survivin. This complex may be involved in the regulation of the balance between cell division and apoptosis.
Insights
Researchers identified Aurora kinase as a novel RasGAP effector protein. This interaction inhibits Aurora kinase activity and suggests a role in regulating cell division and apoptosis balance.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- Ras GTPase-activating protein (RasGAP) regulates Ras proteins, acting as both a negative regulator and effector.
- In tumors, RasGAP's effector function predominates due to its inability to deactivate oncogenic Ras proteins.
Purpose of the Study:
- To identify downstream effectors of RasGAP that mediate its function in tumor cells.
- To investigate the role of RasGAP Src homology 3 (SH3) domain partners in cell proliferation and apoptosis.
Main Methods:
- Coimmunoprecipitation experiments to identify RasGAP SH3 domain-binding proteins.
- Pull-down assays to confirm interactions between RasGAP and human Aurora/Ipl1-related kinases (HsAIRKs).
- Analysis of RasGAP binding to the kinase domain of Aurora and its effect on kinase activity.
Main Results:
- Identification of Aurora, a Drosophila kinase, as a novel RasGAP SH3 domain-binding protein.
- Confirmation of interactions between RasGAP and human orthologs HsAIRK-1 and HsAIRK-2.
- Demonstration that RasGAP binding inhibits HsAIRK-1 and HsAIRK-2 kinase activity.
- Evidence of a ternary complex formation between RasGAP, HsAIRK, and survivin.
Conclusions:
- RasGAP interacts with and inhibits Aurora kinase activity, suggesting a novel effector pathway.
- The RasGAP-Aurora-survivin complex may play a critical role in balancing cell division and apoptosis.
- This finding provides new insights into oncogenic Ras signaling and potential therapeutic targets.