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Regulatory role of the N-terminus of the vacuolar calcium-ATPase in Cauliflower

Malmström, Susanna; Åkerlund, Hans-Erik LU and Askerlund, Per (2000) In Plant Physiology 122(2). p.517-526
Abstract
The vacuolar calmodulin (CaM)-stimulated Ca(2+)-ATPase, BCA1p, in cauliflower (Brassica oleracea) has an extended N terminus, which was suggested to contain a CaM-binding domain (S. Malmström, P. Askerlund, M.G. Palmgren [1997] FEBS Lett 400: 324-328). The goal of the present study was to determine the role of the N terminus in regulating BCA1p. Western analysis using three different antisera showed that the N terminus of BCA1p is cleaved off by trypsin and that the N terminus contains the CaM-binding domain. Furthermore, the expressed N terminus binds CaM in a Ca(2+)-dependent manner. A synthetic peptide corresponding to the CaM-binding domain of BCA1p (Ala-19 to Leu-43) strongly inhibited ATP-dependent Ca(2+) pumping by BCA1p in... (More)
The vacuolar calmodulin (CaM)-stimulated Ca(2+)-ATPase, BCA1p, in cauliflower (Brassica oleracea) has an extended N terminus, which was suggested to contain a CaM-binding domain (S. Malmström, P. Askerlund, M.G. Palmgren [1997] FEBS Lett 400: 324-328). The goal of the present study was to determine the role of the N terminus in regulating BCA1p. Western analysis using three different antisera showed that the N terminus of BCA1p is cleaved off by trypsin and that the N terminus contains the CaM-binding domain. Furthermore, the expressed N terminus binds CaM in a Ca(2+)-dependent manner. A synthetic peptide corresponding to the CaM-binding domain of BCA1p (Ala-19 to Leu-43) strongly inhibited ATP-dependent Ca(2+) pumping by BCA1p in cauliflower low-density membranes, indicating that the CaM-binding region of BCA1p also has an autoinhibitory function. The expressed N terminus of BCA1p and a synthetic peptide (Ala-19 to Met-39) were good substrates for phosphorylation by protein kinase C. Sequencing of the phosphorylated fusion protein and peptide suggested serine-16 and/or serine-28 as likely targets for phosphorylation. Phosphorylation of serine-28 had no effect on CaM binding to the alanine-19 to methionine-39 peptide. Our results demonstrate the regulatory importance of the N terminus of BCA1p as a target for CaM binding, trypsin cleavage, and phosphorylation, as well as its importance as an autoinhibitory domain. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Plant Physiology
volume
122
issue
2
pages
517 - 526
publisher
American Society of Plant Biologists
external identifiers
  • scopus:0034144402
ISSN
1532-2548
DOI
10.1104/pp.122.2.517
language
English
LU publication?
yes
id
f691bca8-e0d3-46a6-94d6-8ca12f9c12b1 (old id 1474798)
date added to LUP
2009-09-29 18:20:02
date last changed
2017-01-01 07:33:22
@article{f691bca8-e0d3-46a6-94d6-8ca12f9c12b1,
  abstract     = {The vacuolar calmodulin (CaM)-stimulated Ca(2+)-ATPase, BCA1p, in cauliflower (Brassica oleracea) has an extended N terminus, which was suggested to contain a CaM-binding domain (S. Malmström, P. Askerlund, M.G. Palmgren [1997] FEBS Lett 400: 324-328). The goal of the present study was to determine the role of the N terminus in regulating BCA1p. Western analysis using three different antisera showed that the N terminus of BCA1p is cleaved off by trypsin and that the N terminus contains the CaM-binding domain. Furthermore, the expressed N terminus binds CaM in a Ca(2+)-dependent manner. A synthetic peptide corresponding to the CaM-binding domain of BCA1p (Ala-19 to Leu-43) strongly inhibited ATP-dependent Ca(2+) pumping by BCA1p in cauliflower low-density membranes, indicating that the CaM-binding region of BCA1p also has an autoinhibitory function. The expressed N terminus of BCA1p and a synthetic peptide (Ala-19 to Met-39) were good substrates for phosphorylation by protein kinase C. Sequencing of the phosphorylated fusion protein and peptide suggested serine-16 and/or serine-28 as likely targets for phosphorylation. Phosphorylation of serine-28 had no effect on CaM binding to the alanine-19 to methionine-39 peptide. Our results demonstrate the regulatory importance of the N terminus of BCA1p as a target for CaM binding, trypsin cleavage, and phosphorylation, as well as its importance as an autoinhibitory domain.},
  author       = {Malmström, Susanna and Åkerlund, Hans-Erik and Askerlund, Per},
  issn         = {1532-2548},
  language     = {eng},
  number       = {2},
  pages        = {517--526},
  publisher    = {American Society of Plant Biologists},
  series       = {Plant Physiology},
  title        = {Regulatory role of the N-terminus of the vacuolar calcium-ATPase in Cauliflower},
  url          = {http://dx.doi.org/10.1104/pp.122.2.517},
  volume       = {122},
  year         = {2000},
}