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Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
Published on: June 25, 2020
Divalent cation effects on interactions between multiple Arabidopsis 14-3-3 isoforms and phosphopeptide targets
Michael S Manak1, Robert J Ferl
1Program in Plant Molecular and Cellular Biology, Horticultural Sciences, University of Florida, 1143 Hull Road, Fifield Hall 110690, Gainesville, Florida 32611-0690, USA.
Biochemistry
|January 24, 2007
Summary
Cellular divalent cations like calcium and magnesium enhance the interaction between 14-3-3 proteins and their target peptides. This binding affinity increases with higher cation concentrations, impacting cellular signaling pathways.
Area of Science:
- Plant Molecular Biology
- Biochemistry
- Cell Signaling
Background:
- Cellular divalent cations, including calcium and magnesium, play crucial roles in signal transduction.
- 14-3-3 proteins are key regulators of cellular processes, interacting with various target proteins.
- Divalent cation binding induces conformational changes in 14-3-3 proteins, increasing their hydrophobicity.
Purpose of the Study:
- To investigate the effect of divalent cations (calcium and magnesium) on the interaction between Arabidopsis 14-3-3 proteins and target peptides.
- To quantify the binding affinity under physiologically relevant cation concentrations.
- To assess this interaction across diverse 14-3-3 isoforms and target peptide classes.
Main Methods:
- Surface Plasmon Resonance (SPR) for real-time binding analysis.
- Isothermal Titration Calorimetry (ITC) for thermodynamic characterization of binding.
- Utilized ten recombinant Arabidopsis 14-3-3 isoforms and two synthetic phosphopeptides (based on NR2 and AHA2).
Main Results:
- Increased concentrations of calcium or magnesium ions consistently enhanced the binding affinity between 14-3-3 proteins and their target phosphopeptides.
- This effect was observed across all tested 14-3-3 isoforms, representing all major phylogenetic branches.
- Binding enhancement was dose-dependent, occurring within physiologically relevant cation concentration ranges (1 µM to 1 mM for Ca2+, 1 µM to 5 mM for Mg2+).
Conclusions:
- Divalent cations are critical modulators of 14-3-3 protein-phosphopeptide interactions.
- The findings highlight a conserved mechanism by which cation levels influence 14-3-3 mediated signaling in plants.
- This cation-dependent regulation provides a dynamic control point for signal transduction pathways involving 14-3-3 proteins.
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