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Calmodulin binds to and inhibits GTP binding of the ras-like GTPase Kir/Gem
1Department of Veterinary Biochemistry, University Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Abstract:
Recently, a new subfamily of Ras-related GTP-binding proteins consisting of Rad (Ras associated with diabetes), Gem (immediate early gene expressed in mitogen-stimulated T-cells), and Kir (tyrosine kinase-inducible Ras-like) was discovered. The C terminus of these proteins contains an extension of approximately 30 amino acids not present in other members of the Ras family and which exhibits all the hallmarks typical for calmodulin (CaM)-binding domains. A peptide corresponding to the putative CaM-binding domain of the Kir/Gem protein was synthesized, and its affinity for CaM was determined by fluorescence spectrometry. Titration of dansyl-CaM with the Kir/Gem peptide gave an affinity constant of 1 nM. Furthermore, a single point mutation of the peptide, W269G, abolished this high affinity interaction. Gel-shift analysis showed that the complex formation between CaM and the Kir/Gem peptide is strictly calcium-dependent. We also demonstrate with a newly developed [32P]CaM overlay technique that full-length Kir/Gem and Rad proteins bind CaM in a Ca2+-dependent fashion. The binding of CaM to glutathione S-transferase-Kir and GST-Gem inhibited the binding of GTP to Kir/Gem significantly. These results suggest the existence of a direct link between Ca2+/CaM and growth factor signal transduction pathways at the level of small Ras-like GTPases.
Insights
A newly discovered Ras-related protein subfamily binds calmodulin (CaM) in a calcium-dependent manner. This interaction inhibits GTP binding, suggesting a link between Ca2+/CaM and growth factor signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- A novel subfamily of Ras-related GTP-binding proteins, including Rad, Gem, and Kir, has been identified.
- These proteins possess a unique C-terminal extension resembling calmodulin (CaM)-binding domains.
Purpose of the Study:
- To investigate the interaction between the Kir/Gem protein's CaM-binding domain and CaM.
- To determine the functional consequences of CaM binding on Ras-related GTPase activity.
Main Methods:
- Synthesis and characterization of a Kir/Gem peptide for CaM-binding studies.
- Fluorescence spectrometry and gel-shift analysis to assess CaM-peptide affinity and calcium dependence.
- Development of a [32P]CaM overlay technique to study CaM binding to full-length proteins.
- Glutathione S-transferase (GST) pull-down assays to evaluate the effect of CaM binding on GTP binding.
Main Results:
- The Kir/Gem peptide exhibits high-affinity binding to CaM (K_a = 1 nM), which is abolished by a single point mutation (W269G).
- CaM-peptide complex formation is strictly calcium-dependent.
- Full-length Kir/Gem and Rad proteins bind CaM in a Ca2+-dependent manner.
- CaM binding to GST-Kir and GST-Gem significantly inhibits their GTP binding.
Conclusions:
- Calmodulin directly binds to a novel subfamily of Ras-related GTP-binding proteins (Rad, Gem, Kir) in a calcium-dependent manner.
- This CaM interaction modulates the GTP-binding activity of these proteins.
- The findings suggest a direct link between Ca2+/CaM and growth factor signal transduction pathways via small Ras-like GTPases.