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Salicylic acid-inducible Arabidopsis CK2-like activity phosphorylates TGA2

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Abstract

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.

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Abbreviations

ASF-1:

Activation Sequence Factor-1

aa:

Amino acid

bZIP:

Basic region/leucine zipper

CK2:

Casein Kinase II

EMSA:

Electrophoretic mobility shift assay

GFP:

Green Fluorescent Protein

PAGE:

Polyacrylamide gel electrophoresis

TF:

Transcription Factor

SARP:

Salicylic Acid Response Protein

wt:

Wild type

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  1. Boyce Thompson Institute for Plant Research, Tower Road, Ithaca NY, 14853, USA

    Hong-Gu Kang & Daniel F. Klessig

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  1. Hong-Gu Kang
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Kang, HG., Klessig, D.F. Salicylic acid-inducible Arabidopsis CK2-like activity phosphorylates TGA2. Plant Mol Biol 57, 541–557 (2005). https://doi.org/10.1007/s11103-005-0409-1

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