Identification of Calmodulin Isoform-specific Binding Peptides from a Phage-displayed Random 22-mer Peptide Library

A tobacco calcium/calmodulin-binding protein kinase (NtCBK1) was isolated and identified. The predicted NtCBK1 protein has 599 amino acids, an N-terminal kinase domain, and shares high homology with other calmodulin (CaM)-related kinases. Whereas NtCBK1 phosphorylates itself and substrates such as histone IIIS and syntide-2 in the absence of CaM, its kinase activity can be stimulated by tobacco CaMs. However, unlike another tobacco protein kinase designated NtCBK2, NtCBK1 was not differentially regulated by the different CaM isoforms tested. The CaM-binding domain of NtCBK1 was located between amino acids 436 and 455, and this domain was shown to be necessary for CaM modulation of kinase activity. RNA in situ hybridization showed that NtCBK1 was highly regulated in the transition to flowering. Whereas NtCBK1 mRNA was accumulated in the shoot apical meristem during vegetative growth, its expression was dramatically decreased in the shoot apical meristem after floral determination, and in young flower primordia. The expression of NtCBK1 was up-regulated to high levels in floral organ primordia. Fluctuations in NtCBK1 expression were verified by analysis of tobacco plants expressing green fluorescent protein under the control of the NtCBK1 promoter, suggesting a role of NtCBK1 in the transition to flowering. This conclusion was confirmed by overexpressing NtCBK1 in transgenic tobacco plants, where maintenance of high levels of NtCBK1 in the shoot apical meristem delayed the switch to flowering and extended the vegetative phase of growth. Further work indicated that overexpression of NtCBK1 in transgenic tobacco did not affect the expression of NFL, a tobacco homologue of the LFY gene that controls meristem initiation and floral structure in tobacco. In addition, the promotion of tobacco flowering time by DNA demethylation cannot be blocked by the overexpression of NtCBK1.

Section: Discussionmentioning

confidence: 99%

A Tobacco Calcium/Calmodulin-binding Protein Kinase Functions as a Negative Regulator of Flowering

Hua

Zhang

Liang

et al. 2004

“…For example, Arabidopsis has 11 CaM genes that encode for seven CaM isoforms. The different CaM isoforms differ in their ability to bind and activate known CaM-regulated enzymes in vitro (57,58). In this study, potato CaM PCM6 and bovine CaMs were used, and no significant differences were found in CaM binding and enzyme activation.…”

Section: Crck1 Is a Cam-binding Cytosolic Protein Kinase In Planta-mentioning

confidence: 96%

Calcium/Calmodulin Up-regulates a Cytoplasmic Receptor-like Kinase in Plants

Yang

Chaudhuri

Yang

et al. 2004

Calcium/calmodulin-dependent kinases play an important role in protein phosphorylation in eukaryotes. However, not much is known about calcium/calmodulin-dependent protein phosphorylation and its role in signal transduction in plants. By using a protein-protein interaction-based approach, we have isolated a novel plantspecific calmodulin-binding receptor-like cytoplasmic kinase (CRCK1) from Arabidopsis thaliana, as well as its ortholog from Medicago sativa (alfalfa). CRCK1 does not show high homology to calcium/calmodulin-dependent protein kinases in animals. In contrast, it shows high homology in the kinase domain to serine/threonine receptor-like kinases in plants. However, it contains neither a transmembrane domain nor an extracellular domain. Calmodulin binds to CRCK1 in a calcium-dependent manner with an affinity of ϳ20.5 nM. The calmodulin-binding site in CRCK1 is located in amino acids 160 -183, which overlap subdomain II of the kinase domain. CRCK1 undergoes autophosphorylation in the presence of Mg 2؉ at the threonine residue(s). The K m and V max values of CRCK1 for ATP are 1 M and 33.6 pmol/mg/min, respectively. Calcium/calmodulin stimulates the kinase activity of CRCK1, which increases the V max of CRCK1 ϳ9-fold. The expression of CRCK1 is increased in response to stresses such as cold and salt and stress molecules such as abscisic acid and hydrogen peroxide. These results indicate the presence of a calcium/calmodulin-regulated receptor-like cytoplasmic kinase in plants. Furthermore, these results also suggest that calcium/calmodulin-regulated protein phosphorylation involving CRCK1 plays a role in stress signal transduction in plants.

“…1B) (37). Moreover, the C-terminal region of the protein (amino acids 726 -845) contains a cluster of several putative CaM-binding domains, four Ca 2ϩ -dependent CaM dissociation domains (IQ motifs), and a Ca 2ϩ -dependent CaM-binding domain (38,39). These structural features suggest that this clone is a transcription factor whose activity can be modulated by CaM binding.…”

Section: Molecular Cloning Of a Cdna Encoding A Cam-binding Transcripmentioning

confidence: 99%

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

Choi

Kim

Yoo

et al. 2005

Self Cite

121

128

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:horseradish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-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 Ca 2؉ -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 ␤-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.