Effects of interaction with calcineurin on the reactivities of calmodulin lysines.
Calmodulin was trace labeled by acetylation with [3H]acetic anhydride in the presence and absence of a 30% molar excess of the phosphatase calcineurin; phenylalanine was included in the reaction mixtures as an internal standard. The level of 3H acetylation of each of the 7 lysines was determined and corrected for differences arising from reaction conditions using the labeling of the internal standard, following procedures that are closely similar to those used in a previous study of the interaction of calmodulin with myosin light chain kinase (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232). The interaction with calcineurin was found to produce a 10-fold reduction in the acetylation of lysine 75, with lesser but significant effects on lysines 21 and 148. A small but reproducible perturbation of lysine 77 was also observed. The results are similar to those that are produced by the interaction with myosin light chain kinase. However, when they are compared with two recent reports between which there are major discrepancies (Manalan, A. S., and Klee, C. B. (1987) Biochemistry 26, 1382-1390; Winkler, M. A., Fried, V. A., Merat, D. L., and Cheung, W. Y. (1987) J. Biol. Chem. 262, 15466-15471), our results are in good agreement with those obtained in the former study. From the location of the perturbed groups in the three-dimensional structure of calmodulin, it appears that the interaction site on calmodulin for calcineurin, as well as for myosin light chain kinase, is very extended and may include hydrophobic pockets at homologous sites near the carboxyl-terminal ends of the two halves of the molecule.
Wei
Q
Q
Department of Biochemistry and Molecular Biology, University of Miami, Florida 33136.
Jackson
A E
AE
Pervaiz
S
S
Carraway
K L
KL
Lee
E Y
EY
Puett
D
D
Brew
K
K
D000089:Acetic Anhydrides; D000107:Acetylation; D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002132:Calcium Radioisotopes; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D008239:Lysine; D010649:Phenylalanine; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D014316:Tritium
D000089:Acetic Anhydrides; D002132:Calcium Radioisotopes; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D014316:Tritium; C031800:acetic anhydride; D010649:Phenylalanine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D008239:Lysine