Regulatory role of the N terminus of the vacuolar calcium-ATPase in cauliflower.
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.
Malmström
S
S
Department of Plant Biochemistry, Lund University, P.O. Box 117, SE-221 00 Lund, Sweden. susanna.malmstrom@plantbio.lu.se
Akerlund
H E
HE
Askerlund
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D017931:DNA Primers; D017136:Ion Transport; D008969:Molecular Sequence Data; D010766:Phosphorylation; D018517:Plant Roots; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology*
D002147:Calmodulin; D017931:DNA Primers; D012333:RNA, Messenger; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; D002118:Calcium