Phosphorylation of the calmodulin binding domain of the plasma membrane Ca2+ pump by protein kinase C reduces its interaction with calmodulin and with its pump receptor site

• Published in: The Journal of biological chemistry 1994-09, Vol.269 (39), p.24298-24303
• Main Authors: Hofmann, F, Anagli, J, Carafoli, E, Vorherr, T
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Calcium-Transporting ATPases - metabolism; Calmodulin - metabolism; Calmodulin-Binding Proteins - metabolism; Erythrocyte Membrane - metabolism; Humans; Molecular Sequence Data; Phosphorylation; Protein Binding; Protein Kinase C - metabolism; Threonine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)51081-X

Two versions of the calmodulin binding domain of the plasma membrane Ca2+ ATPase, a 24-amino acid peptide, C24W (Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-R-V-V-N-A-F-R-S-S-NH2), and the corresponding phosphothreonine containing peptide, C24W-P (Q-I-L-W-F-R-G-L-N-R-I-Q-T(phospho)-Q-I-R-V-V-N-A-F-R-S-S-NH2), were synthesized. They were used to investigate the effect of threonine phosphorylation by protein kinase C on the binding of calmodulin by the calmodulin binding domain and on the inhibitory role of the domain on the activity of the Ca2+ pump. The phosphopeptide C24W-P was obtained after global phosphorylation of the free Thr side chain on the protected resin bound peptide. The phosphorylated calmodulin binding domain failed to bind calmodulin; this was shown by gel shift experiments, by fluorescence energy transfer studies and by competition experiments against calmodulin stimulation of the pump. The inhibition of the Ca2+ pump activity by the calmodulin binding domain in the absence of calmodulin was also affected by the phosphorylation of the threonine; the inhibition of the fully active calpain-truncated pump by the phosphothreonine containing peptide was lower than that by the unphosphorylated synthetic domain.