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A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains
Year:
2000
Source of publication :
Plant Molecular Biology
Authors :
Arazi, Tzahi
;
.
Volume :
42
Co-Authors:
Arazi, T., Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel
Kaplan, B., Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel
Fromm, H., Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel, Centre for Plant Sciences, Leeds Inst. for Biotech. and Agric., University of Leeds, Leeds LS2 9JT, United Kingdom
Facilitators :
From page:
591
To page:
601
(
Total pages:
11
)
Abstract:
Recently we isolated a cDNA encoding a tobacco plasma membrane calmodulin-binding channel protein (designated NtCBP4) with a putative cyclic nucleotide-binding domain. Here we analyzed in detail the interaction of NtCBP4 with calmodulin. A full-length recombinant NtCBP4 (81 kDa) expressed in Sf9 insect cells, and the corresponding tobacco membrane protein were solubilized from their respective membrane fractions and partially purified by calmodulin affinity chromatography. NtCBP4 was detected in the eluted fractions using specific antibodies raised against the recombinant protein. By binding [35S]-calmodulin to recombinant NtCBP4 truncations fused to glutathione S-transferase, we identified a single region consisting of 66 amino acids capable of binding calmodulin. A 23 amino acid synthetic peptide from within this region formed a complex with calmodulin in the presence of calcium. We measured the fluorescence of dansyl-calmodulin interacting with this peptide, which revealed a dissociation constant of about 8 nM. The NtCBP4 calmodulin-binding domain was found to perfectly coincide with a phylogenetically conserved αC-helix motif of its putative cyclic nucleotide-binding domain. Furthermore, a 23 amino acid region in an equivalent site in the cAMP-binding domain of a mammalian protein kinase regulatory subunit was also found to bind calmodulin. Thus, coinciding calmodulin- and cyclic nucleotide-binding domains may serve as a point of communication between calcium and cyclic nucleotide signal transduction pathways in plants and animals.
Note:
Related Files :
Animals
CaM-binding channel
cAMP-dependent kinase
cattle
Cyclic AMP
Mammalia
Plants, Toxic
solubilization
Show More
Related Content
More details
DOI :
10.1023/A:1006345302589
Article number:
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
31216
Last updated date:
02/03/2022 17:27
Creation date:
17/04/2018 01:00
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Scientific Publication
A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains
42
Arazi, T., Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel
Kaplan, B., Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel
Fromm, H., Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel, Centre for Plant Sciences, Leeds Inst. for Biotech. and Agric., University of Leeds, Leeds LS2 9JT, United Kingdom
A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains
Recently we isolated a cDNA encoding a tobacco plasma membrane calmodulin-binding channel protein (designated NtCBP4) with a putative cyclic nucleotide-binding domain. Here we analyzed in detail the interaction of NtCBP4 with calmodulin. A full-length recombinant NtCBP4 (81 kDa) expressed in Sf9 insect cells, and the corresponding tobacco membrane protein were solubilized from their respective membrane fractions and partially purified by calmodulin affinity chromatography. NtCBP4 was detected in the eluted fractions using specific antibodies raised against the recombinant protein. By binding [35S]-calmodulin to recombinant NtCBP4 truncations fused to glutathione S-transferase, we identified a single region consisting of 66 amino acids capable of binding calmodulin. A 23 amino acid synthetic peptide from within this region formed a complex with calmodulin in the presence of calcium. We measured the fluorescence of dansyl-calmodulin interacting with this peptide, which revealed a dissociation constant of about 8 nM. The NtCBP4 calmodulin-binding domain was found to perfectly coincide with a phylogenetically conserved αC-helix motif of its putative cyclic nucleotide-binding domain. Furthermore, a 23 amino acid region in an equivalent site in the cAMP-binding domain of a mammalian protein kinase regulatory subunit was also found to bind calmodulin. Thus, coinciding calmodulin- and cyclic nucleotide-binding domains may serve as a point of communication between calcium and cyclic nucleotide signal transduction pathways in plants and animals.
Scientific Publication
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