Marie H. Krinks

Factors responsible for the Ca2+-dependent inactivation of calcineurin in brain

The Ca2+‐dependent protein phosphatase activity of crude rat brain extracts measured in the presence of okadaic acid, exhibits the characteristic properties of the calmodulin‐stimulated protein phosphatase, calcineurin. It is stimulated more than 200‐fold by Ca2+ and inhibited by the calmodulin‐binding peptide, M13, and by the immunosuppressive drug, FK506. It is insensitive to rapamycin at concentrations up to 1 μM. Its specific activity, based on calcineurin concentration determined by quantitative analysis of Western blots exposed to anti‐bovine brain IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions. Unlike the purified enzyme it is rapidly and irreversibly inactivated in a time‐, temperature‐, and Ca2+/calmodulin‐dependent fashion without evidence of extensive proteolytic degradation. The enzyme is converted to a state which does not lose activity by removal of low molecular weight material by gel filtration. Reconstitution of a labile enzyme is achieved by the addition of the low molecular weight‐containing fraction eluted from the gel filtration column. These observations indicate that calcineurin in crude brain extracts is under the control of Ca2+/calmodulin‐dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).

Heteronuclear 3D NMR and isotopic labeling of calmodulin. Towards the complete assignment of the 1H NMR spectrum.

New methods are described that permit detailed analysis of the NMR spectra of calmodulin, an alpha-helical protein with a molecular weight of 16.7 kD. Two complementary approaches have been used: uniform labeling with 15N and labeling of specific amino acids with either 15N or 13C. It is demonstrated that uniform 15N labeling permits the recording of sensitive three-dimensional (3D) NMR spectra that show far better resolution than their conventional two-dimensional analogs. Selective 15N labeling of amino acids can be used for identifying the type of amino acid, providing information that is essential for the analysis of the 3D spectra. Simultaneous selective labeling with both 15N and 13C can provide a number of unique backbone assignments from which sequential assignment can be continued.

An efficient NMR approach for obtaining sequence-specific resonance assignments of larger proteins based on multiple isotopic labeling.

By simultaneously incorporating in a protein 13C‐carbonyl‐ and 15N‐labeled amino acids with different levels of enrichment, characteristic asymmetric doublet‐like patterns are observed for 15N nuclei that are directly adjacent to the 13C1‐labeled residues, providing unambiguous identification of a large number of unique dipeptide fragments of the protein. Additional assignments and qualitative structural information can be obtained from such a selectively labeled protein by recording multiple bond correlation spectra. The procedure is demonstrated for the protein calmodulin, complexed with calcium.

Calcineurin: A Member of a Family of Calmodulin-Stimulated Protein Phosphatases

Abstract Calcineurin, a major calmodulin-binding protein of brain, is a heterodimer composed of a 61,000 M r calmodulin-binding subunit, calcineurin A, and a 19,000 M r Ca2+-binding subunit, calcineurin B. The discovery of a calmodulin-regulated protein phosphatase in rabbit skeletal muscle with a similar subunit structure led to the identification of calcineurin as a protein phosphatase (AA Stewart, TS Ingebritsen, A Manalan, CB Klee, P Cohen (1982) FEBS Lett 137:80–84). Using rabbit polyclonal antibodies to bovine brain calcineurin, both subunits of calcineurin can be identified in crude homogenates of bovine brain by an immunoblotting technique. In crude homogenates of bovine skeletal and cardiac muscle, a 59,000–61,000 M r doublet and a 15,000 M r species (the electrophoretic mobility of calcineurin B) are also detected by this technique. The cross-reactivity of these species with antibodies to brain calcineurin indicates antigenic similarity between the muscle proteins and calcineurin, and suggests the existence of a family of structurally related calmodulin-stimulated protein phosphatases. Like calcineurin, the 61,000 M r subunits in skeletal and cardiac muscle bind calmodulin and are detected in crude tissue extracts by 125I-calmodulin gel overlay. Thus, both the 125I-calmodulin gel overlay method and the immunoblotting technique are useful in screening crude preparations, in which detection of calmodulin-stimulated protein phosphatase activity may be complicated by the many phosphatases present.

Activation of enzymes by calmodulins containing intramolecular cross-links.

We have reacted calmodulins containing cysteines substituted at positions 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate intramolecularly cross-linked proteins termed BMHCM or BMHCM1, respectively. Reactions were also performed with N-ethylmaleimide (NEM) in place of BMH to generate corresponding S-ethylsuccinimidylated proteins termed NEMCM or NEMCM1. The abilities of these proteins to activate plant NAD kinase, erythrocyte Ca(2+)-ATPase and bovine brain calcineurin activities were assessed. The BMH- or NEM-reacted proteins activate calcineurin activity as does control calmodulin. Kact values for Ca(2+)-ATPase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in Vmax values. Activation of this enzyme by NEMCM or NEMCM1 is not different from the control. In NAD kinase activation experiments BMHCM and BMHCM1 are associated with a 10 to 20-fold increase in Kact values and a 60-75% reduction in Vmax values relative to the control. NEMCM1 is not associated with any apparent changes in NAD kinase activation, however, NEMCM is associated with a 10-fold increase in the Kact value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased Kact value, implying that this change is due to interactions between S-(ethylsuccinimido)cysteines at positions 3 and 146. In conclusion, cross-linking and associated distortions in the structure of calmodulin appear to have little or no effect on activation of calcineurin enzyme activity. However, bending in the central helix and/or steric restrictions associated with cross-linking increase significantly the Kact value for Ca(2+)-ATPase and NAD kinase activation, and dramatically reduce maximal activation of NAD kinase activity.

THE DOMAIN STRUCTURE OF CALCINEURIN

Publisher Summary This chapter discusses the domain structure of calcineurin. A calmodulin-stimulated protein phosphatase was first isolated from skeletal muscle and was also shown to be associated with calcineurin, the major calmodulin and Ca 2+ -binding protein of brain extracts. Calmodulin-stimulated phosphatases have been detected in many mammalian tissues. Most, like calcineurin, appear to be composed of two subunits, calcineurin A and calcineurin B, and cross react with polyclonal antibodies to bovine brain calcineurin. The antibodies also recognize calcineurin B-like proteins in several lower eukaryotes such as Paramecium tetraurelia, Drosophila melanogaster, and sea urchins but cross react more poorly with the large subunit in invertebrates and vertebrate tissues other than brain. Calmodulin-stimulated protein phosphatase has a wide tissue and species distribution, but its large subunit may exist as different isozymes. Brain, which contains 10–20 fold more of this enzyme than other tissues, is the richest source of the protein. The affinity of calcineurin for calmodulin is one of the highest reported for calmodulin stimulated enzymes. It is found that calmodulin does not compete with calcineurin B for the calcineurin B binding site on calcineurin A in reconstitution experiments.