Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation.
The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10-20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.
Maréchal
E
E
Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10021-6399, USA.
Hiratsuka
K
K
Delgado
J
J
Nairn
A
A
Qin
J
J
Chait
B T
BT
Chua
N H
NH
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010947:Plants, Toxic; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C077926:GT-1 transcription factor; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium