Functional domain structure of calcineurin A: mapping by limited proteolysis
- 21 February 1989
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 28 (4) , 1868-1874
- https://doi.org/10.1021/bi00430a066
Abstract
Limited proteolysis of calcineurin, the Ca2+/calmodulin-stimulated protien phosphatase, with clostripain is sequential and defines four functional domains in calcineurin A (61 kDa). In the presence of calmodulin, an inhibitory domain located at the carboxyl terminus is rpadily degraded, yielding an Mr 57 000 fragment which retains the ability to bind calmodulin but whose p-nitrophenylphosphatase is fully active in the absence of Ca2+ and no longer stimulated by calmodulin. Subsequent cleavage(s), near the amino terminus, yield(s) and Mr 55 000 fragment which has lost more than 80% of the enzymatic activity. A third, slower, proteolytic cleavage in the carboxyl-terminal half of the protein converts the Mr 55 000 fragment to an Mr 42 000 polypeptide which contains the calcineurin B binding domain and an Mr 14 000 fragment which binds calmodulin in a Ca2+-dependent manner with high affinity. In the absence of calmodulin, clostripain rapidly severs both the calmodulin-binding and the inhibitory domains. The catalytic domain is preserved, and the activity of the proteolyzed 43-kDa enzyme is increased 10-fold in the absence of Ca2+ and 40-fold in its presence. The calcineurin B binding domain and calcineurin B appear unaffected by proteolysis both in the presence and in the absence of calmodulin. Thus, calcineurin A is organized into functionally distinct domains connected by proteolytically sensitive hinge regions. The catalytic, inhibitory, and calmodulin-binding domain are readily removed from the protease-resistant core, which contains the calcineurin B binding domain. Calmodulin stimulation of calcineurin is dependent on intact inhibitory and calmodulin-binding domains, but the degraded enzyme lacking these domains is still regulated by Ca2+.This publication has 30 references indexed in Scilit:
- The structure of the B subunit of calcineurinEuropean Journal of Biochemistry, 1984
- Activation of bovine brain calmodulin-dependent protein phosphatase by limited trypsinizationBiochemistry, 1984
- Amino acid analysis by reverse-phase high-performance liquid chromatography: Precolumn derivatization with phenylisothiocyanateAnalytical Biochemistry, 1984
- Activation of calcineurin by limited proteolysis.Proceedings of the National Academy of Sciences, 1983
- Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density.The Journal of cell biology, 1981
- Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells.Journal of Biological Chemistry, 1980
- Calcineurin: a calcium- and calmodulin-binding protein of the nervous system.Proceedings of the National Academy of Sciences, 1979
- Conformational transition accompanying the binding of calcium(2+) ion to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesteraseBiochemistry, 1977
- A spectrophotometric determination of trypsin and chymotrypsinBiochimica et Biophysica Acta, 1955
- PREPARATION AND MEASUREMENT OF THE PURITY OF THE PHOSPHATASE REAGENT, DISODIUM p-NITROPHENYL PHOSPHATEJournal of Biological Chemistry, 1952