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Abstract

HBP-1a(17) is representative of a group of plant bZIP-type transcription factors which includes HBP-1a proteins and G-box-binding factors. We found kinase activity in wheat nuclear extract that phosphorylated HBP-1a(17). Experiments using recombinant HBP-1a(17) derivatives as substrates revealed that all three of the Ser residues in the basic region, Ser-261, Ser-265, and Ser-269, were phosphorylated in a Ca(2+)-stimulated manner. DNA-binding analysis of mutants with a Ser-to-Glu change, prepared to mimic the phosphorylated proteins, indicated that introduction of a negative charge at position 265 or 269 prevents HBP-1a(17) from binding DNA not only in the homodimer of mutants but also in heterodimers with a wild-type protein. It is therefore suggested that the phosphorylation regulates the function of HBP-1a(17) at least at the level of DNA binding. Since Ser-265 and Ser-269 are highly conserved among the plant bZIP-type factors known to date, a common Ca(2+)-mediated regulatory mechanism may exert an effect on the bZIP-type factors through phosphorylation of these conserved Ser residues.

Phosphorylation of the Parsley bZIP Transcription Factor CPRF2 Is Regulated by Light

“…Expression and Purification of Recombinant CPRF2-The restriction fragment encoding full-length CPRF2 was subcloned into the BamHI site of the pQE70 vector (Qiagen) to produce a fusion protein with a C-terminal (His) 6 tag. Transformation of the vectors in Escherichia coli, expression and purification of the proteins on nickel nitrilotriacetic acid (Ni-NTA)-agarose were performed under denaturating conditions as described in the manufacturer's protocol (Qiagen).…”

“…1 min after addition of 5 Ci of [␥- 32 P]ATP to the samples the reaction was stopped with 500 l of 6 M guanidine hydrochloride, 0.1 M NaH 2 PO 4 , 0.01 M Tris, pH 8.0. The (His) 6 -tagged proteins were isolated on Ni-NTA-agarose, eluted with 50 l of 8 M urea, 0.1 M NaH 2 PO 4 , 0.01 M Tris, pH 6.3, and 100 mM EDTA and tested for [ 32 P]phosphate incorporation as described above. Silver staining of Ni-NTA purified CPRF2 was performed according to Ref.…”

“…As shown, for example, for chalcone synthase or chlorophyll a/b-binding protein genes, promoter elements that mediate light responsiveness frequently contain the palindromic DNA motif ACGT, that, depending on the adjacent nucleotides, is part of the so-called G-box (CACGTG) or C-box (GACGTC) sequences (5). However, G-and C-boxes are not only found in the promoters of lightregulated genes but also in promoters of genes that respond to other exogenic and endogenic stimuli such as stress, hormones, and cell cycle-related signals (6). Transcription factors containing a basic leucine-zipper motif (bZIP), 1 as, for example, the common plant regulatory factors (CPRFs) from parsley (7)(8)(9)(10)(11) and G-box binding factors from Arabidopsis (12)(13)(14), were shown to bind to the G-box or the C-box, respectively, in vitro as well as in vivo and form specific homo-and heterodimers (7,8,12,13).…”

HRT, a Novel Zinc Finger, Transcriptional Repressor from Barley

“…Many zinc finger proteins are of the cluster type containing multiple tandemly repeated fingers separated by a conserved short sequence. However, several zinc finger proteins, in which the fingers are separated by much longer spacers of various lengths, have been described from plants (46). The spacing between the three fingers in HRT is also wide.…”

ABFs, a Family of ABA-responsive Element Binding Factors

“…Recently, a family of embryospecific bZIP proteins has been reported that can recognize both G/ABRE and C/ABRE (14,15). Also, a whole array of other factors that can bind to the ACGT-containing sequences has been described (16,17).…”