Isolation of a Calmodulin-binding Transcription Factor from Rice (Oryza sativa L.)

• Published in: The Journal of biological chemistry 2005-12, Vol.280 (49), p.40820-40831
• Main Authors: Choi, Man Soo, Kim, Min Chul, Yoo, Jae Hyuk, Moon, Byeong Cheol, Koo, Sung Cheol, Park, Byung Ouk, Lee, Ju Huck, Koo, Yoon Duck, Han, Hay Ju, Lee, Sang Yeol, Chung, Woo Sik, Lim, Chae Oh, Cho, Moo Je
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arabidopsis; Arabidopsis - genetics; Base Sequence; Binding Sites; Calmodulin - genetics; Calmodulin-Binding Proteins - chemistry; Calmodulin-Binding Proteins - genetics; Calmodulin-Binding Proteins - isolation & purification; Cloning, Molecular; DNA, Complementary - genetics; DNA, Plant - chemistry; DNA, Plant - metabolism; Escherichia coli - genetics; Gene Deletion; Gene Expression; Gene Library; Glucuronidase - genetics; Glutathione Transferase - genetics; Green Fluorescent Proteins - genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oryza - chemistry; Oryza - genetics; Oryza sativa; Promoter Regions, Genetic - genetics; Recombinant Fusion Proteins - genetics; Sequence Alignment; Simian virus 40; Tandem Repeat Sequences; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M504616200

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativaCaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca2+-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5′-TWCG(C/T)GTKKKKTKCG-3′ (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate β-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.