Salicylic acid-inducible Arabidopsis CK2-like activity phosphorylates TGA2.
We demonstrate that TGA2, TGA5 and TGA6, and TGA3 to a lesser extent, are phosphorylated by an activity in rabbit reticulocytes. Using deletion and point mutagenesis of TGA2, three amino acid (aa) residues, (11)Ser, (12)Thr and (16)Thr, were found to be critical for efficient phosphorylation by a kinase(s) in rabbit reticulocytes. These three residues also were important for phosphorylation by recombinant human Casein Kinase II (CK2) and by a CK2-like kinase in Arabidopsis leaf extracts. Salicylic acid (SA) treatment enhanced the phosphorylation of recombinant TGA2 in vitro; it also enhanced phosphorylation of a TGA2-GFP fusion protein in vivo. By contrast, in vivo phosphorylation of a TGA2-A-GFP fusion protein, in which the (11)Ser, (12)Thr and (16)Thr residues were mutated to non-phosphorylable alanine, was only poorly if at all stimulated by SA treatment. Mutation of the putative CK2 phosphorylation motif did not affect nuclear localization of TGA2. However, the DNA binding activity of TGA2 was reduced by CK2 treatment, whereas that of TGA2-A was unaffected; TGA2's DNA binding activity after incubation in a rabbit reticulocyte lysate also was substantially lower than that of comparably treated TGA2-A. Taken together, these results suggest that phosphorylation at the putative CK2 phosphorylation site negatively regulates the DNA binding activity of TGA2. Analysis of transgenic Arabidopsis overexpressing TGA2-GFP or TGA2-A-GFP, in the absence of SA treatment, revealed that they accumulated similarly elevated levels of PR-1 gene transcripts. Possible reasons why mutations in the putative CK2 phosphorylation site had little effect on PR-1 induction by TGA2 are discussed.
Kang
Hong-Gu
HG
Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA.
Klessig
Daniel F
DF
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites / Q000235:genetics; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D030821:Plants, Genetically Modified; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C489413:AHBP-1b protein, Arabidopsis; D029681:Arabidopsis Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C077262:PR-1 protein, Arabidopsis; D047390:Casein Kinase II; D020156:Salicylic Acid