Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation

Area of Science:

• Plant molecular biology
• Gene regulation
• Signal transduction

Background:

• The pea rbcS-3A promoter's BoxII element is crucial for light-mediated gene expression.
• GT-1, a DNA-binding protein interacting with BoxII, is a potential light-modulated molecular switch.
• Light-induced post-translational modifications, like phosphorylation, are hypothesized to alter GT-1 activity.

Purpose of the Study:

• To investigate the role of GT-1 phosphorylation in light-responsive gene activation.
• To identify specific phosphorylation sites on GT-1 and their impact on DNA binding.
• To elucidate the mechanism by which light signals modulate GT-1 activity.

Main Methods:

• In vitro phosphorylation of recombinant Arabidopsis GT-1 (hGT-1) using various kinases.
• Assessing hGT-1 DNA-binding activity after kinase treatment.
• Mass spectrometry to identify phosphorylated residues on hGT-1.
• Site-directed mutagenesis of potential phosphorylation sites and analysis of mutant DNA-binding activity.
• In vitro dephosphorylation assays using calf intestine phosphatase.

Main Results:

• Phosphorylation of hGT-1 by calcium/calmodulin kinase II (CaMKII) increased its DNA-binding activity 10-20 fold.
• Mass spectrometry identified T133 and S198 as heavily phosphorylated residues.
• Mutational analysis revealed that phosphorylation at T133 is primarily responsible for CaMKII-induced DNA-binding enhancement.
• Nuclear GT-1 DNA-binding activity was reduced by phosphatase treatment in light-grown, but not etiolated, plant extracts.

Conclusions:

• GT-1 functions as a molecular switch regulated by calcium-dependent phosphorylation and dephosphorylation.
• Light signals likely modulate GT-1 activity through post-translational modifications, specifically phosphorylation at T133.
• This phosphorylation event enhances GT-1's interaction with the BoxII element, contributing to light-responsive gene expression.