Abstract
The carboxyl-terminal region of the plasma membrane Ca2+ pump isoform 4b contains two autoinhibitory regions which keep the pump inactive in the absence of activators such as calmodulin. One of these regions is approximately coterminous with the calmodulin-binding domain, while the second region is downstream (Verma, A. K., Enyedi, A., Filoteo, A. G., and Penniston, J. T. (1994) J. Biol. Chem. 269, 1687-1691). The carboxyl-terminal region has also been identified as the site for phosphorylation of this isoform by protein kinase C (Wang, K. K. W., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085). Using constructs lacking various numbers of residues at the carboxyl terminus, we studied the degree of phosphorylation by protein kinase C and the resultant activation of Ca2+ transport. The results showed that the most specific and easy phosphorylation occurred in a region of about 20 residues which is downstream of the calmodulin-binding domain, and that the downstream inhibitory domain had also about the same size and location. Phosphorylation partially activated the pump by removing only the inhibition due to this region. Binding of calmodulin to the calmodulin-binding domain activated the pump more fully by removing the inhibition due to both regions, regardless of the state of phosphorylation at the downstream inhibitory region.
Highlights
The plasma membrane Ca2ϩ pump is an important element in regulating the intracellular Ca2ϩ concentration, which removes the excess Ca2ϩ from cells during intracellular Ca2ϩ signaling
The phosphorylation and activation of the plasma membrane Ca2ϩ pump with protein kinase C has been demonstrated both in vivo and in vitro
The phosphorylation of the erythrocyte pump was shown to occur in the carboxyl terminus in a pioneering study by Wang et al [13], who inferred that threonine 1102, in the middle of the calmodulin-binding region, was one of the sites of phosphorylation, while the other was a serine downstream of the calmodulinbinding region
Summary
The plasma membrane Ca2ϩ pump is an important element in regulating the intracellular Ca2ϩ concentration, which removes the excess Ca2ϩ from cells during intracellular Ca2ϩ signaling. That study used calpain digestion of the phosphorylated Ca2ϩ pump and phosphorylation of a synthetic peptide to suggest that one of the phosphorylation sites could be the threonine residue in the middle of the calmodulin-binding domain. In a subsequent study [14], a synthetic peptide representing the calmodulin-binding domain phosphorylated at the threonine in question was studied. Wang et al [13], on the other hand, found that purified type III protein kinase C antagonized the calmodulin activation at low Ca2ϩ concentrations These conflicting results could have various causes, such as the use of different Ca2ϩ pump preparations, different conditions, and/or different protein kinase C isoforms and concentrations, or they might arise from the attempt to compare data on a short peptide with data on the intact pump. The longest construct (ct48) had, in the absence of calmod-
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