A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains

A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains

Loading next page...
 
/lp/springer_journal/a-high-affinity-calmodulin-binding-site-in-a-tobacco-plasma-membrane-0ee06AdvP7
Publisher
Kluwer Academic Publishers
Copyright
Copyright © 2000 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1006345302589
Publisher site
See Article on Publisher Site

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.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 16, 2004

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off