Claude B. Klee

Regulation of the calmodulin-stimulated protein phosphatase, calcineurin.

The role of protein phosphatases in the regulation of cellular processes is now well established (1, 2). Calcineurin (also called protein phosphatase 2B), a major calmodulin-binding protein in brain and the only serine/threonine protein phosphatase under the control of Ca/calmodulin, plays a critical role in the coupling of Ca signals to cellular responses (3–6). Its stimulation by the multifunctional protein, calmodulin, ensures the coordinated regulation of its protein phosphatase activity with the activities of the many other enzymes, including a large number of protein kinases, under Ca and calmodulin control. Despite its special abundance in neural tissues, calcineurin is broadly distributed, and its structure is highly conserved from yeast to man (6). Its resistance to the endogenous phosphatase inhibitor 1 and inhibitor 2 and to the potent inhibitors of protein phosphatase 1 and 2A, okadaic acid, calyculin, and microcystin (1, 2) made it difficult to identify its functions until it was identified as the target of the immunosuppressive drugs, FK506 and cyclosporin A (CsA). Calcineurin was thus shown to play an essential role in T cell activation (7). The demonstration that FK506 and CsA, when bound to their respective binding proteins, FKBP12 and cyclophilin A, are specific inhibitors of calcineurin provided the tools needed to reveal its many other roles in the transduction of Ca signals (8). Its calmodulin dependence distinguishes it from two other known Ca-regulated protein phosphatases, the insulin-sensitive pyruvate dehydrogenase phosphatase of mitochondria (9) and a family of protein phosphatases homologous to the product of the Drosophila retinal degeneration C (rdgC) gene (10–12).

Solution structure of calcium-free calmodulin.

The three-dimensional structure of calmodulin in the absence of Ca2+ has been determined by three- and four-dimensional heteronuclear NMR experiments, including ROE, isotope-filtering combined with reverse labelling, and measurement of more than 700 three-bond J-couplings. In analogy with the Ca2+-ligated state of this protein, it consists of two small globular domains separated by a flexible linker, with no stable, direct contacts between the two domains. In the absence of Ca2+, the four helices in each of the two globular domains form a highly twisted bundle, capped by a short anti-parallel β-sheet. This arrangement is qualitatively similar to that observed in the crystal structure of the Ca2+-free N-terminal domain of troponin C.

Discovery of A Ca2+-and calmodulin-dependent protein phosphatase

Incubation of phosphorylase kinase from rabbit skeletal muscle with cyclic AMP-dependent protein kinase and Mg-ATP causes a rapid phosphorylation of one serine residue on the P-subunit, followed by a phosphorylation of a further serine residue on the a-subunit [l-3]. The activation of phosphorylase kinase which accompanies this reaction is determined solely by the phosphorylation of the P-subunit [3-51. Nevertheless, the serine residue on the a-subunit, as well as that on the@subunit, becomes phosphorylated in vivo in response to adrenaline [6], suggesting that it may have a physiological function. Several years ago we reported that extracts of rabbit skeletal muscle contained two different enzymes which dephosphorylated the P-subunit and a-subunit relatively specifically [7]. These enzymes were initially termed fl-phosphorylase kinase phosphatase and a-phosphorylase kinase phosphatase [7], but were subsequently renamed protein phosphatase-1 and protein phosphatase-2 [3]. Protein phosphatase-2 which had been purified several hundred fold was reported to dephosphorylate histones HI and H2b at similar rates to the a-subunit, and it also contained some glycogen synthase phosphatase and phosphorylase phosphatase activity [8]. Subsequently this enzyme was purified 2500-fold and shown to dephosphorylate a protein termed inhibitor-l [93, which is a potent inhibitor of protein phosphatase-1 [3].

Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3': 5'-monophosphate-dependent protein kinase.

Turkey gizzard smooth muscle light chain kinase was purified by affinity chromatography on calcium dependent regulator weight of 125,000 +/- 5,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When myosin light chain kinase is incubated with the catalytic subunit of cyclic AMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of myosin kinase. Brief tryptic digestion of the 32P-labeled myosin kinase liberates a single radioactive peptide with a molecular weight of approximately 22,000. Phosphorylation of myosin kinase results in a 2-fold decrease in the rate at which the enzyme phosphorylates the 20,000-dalton light chain of smooth muscle myosin. These results suggest that cyclic AMP has a direct effect on actin-myosin interaction in smooth muscle.

Solution structure of Ca(2+)-calmodulin reveals flexible hand-like properties of its domains.

The solution structure of Ca2+-ligated calmodulin is determined from residual dipolar couplings measured in a liquid crystalline medium and from a large number of heteronuclear J couplings for defining side chains. Although the C-terminal domain solution structure is similar to the X-ray crystal structure, the EF hands of the N-terminal domain are considerably less open. The substantial differences in interhelical angles correspond to negligible changes in short interproton distances and, therefore, cannot be identified by comparison of NOEs and X-ray data. NOE analysis, however, excludes a two-state equilibrium in which the closed apo conformation is partially populated in the Ca2+-ligated state. The difference between the crystal and solution structures of Ca2+–calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets. In contrast, the vast majority of side chains making up the target binding surface are locked into the same χ1 rotameric states as in complexes with target peptide.

Calcineurin : from structure to function

Publisher Summary The use of the calcineurin inhibitors—namely, FK506 and CsA, together with yeast genetics and the overexpression of calcineurin by transgenic mice, has established the critical roles of calcineurin in the regulation of many cellular processes that are induced by changes in the concentration of intracellular Ca 2+ in response to external signals. None of the physiological roles of calcineurin is better documented than the regulation of gene expression mediated by the broadly distributed NFAT family of transcription factors in mammalian cells. Equally well documented is the role of calcineurin in the regulation of expression of many genes that are under the control of the TCNI/CRZl transcription factor in yeast. Calcineurim was originally identified as a major calmodulin-binding protein in the brain and later shown to be the only Ca 2+ /calmodulin-regulated serine/threonine protein phosphatase. Since then, this enzyme has been shown to be expressed in every tissue and to be highly conserved phylogenetically.

Identification of the NH2-terminal blocking group of calcineurin B as myristic acid

The NH2‐terminal blocking group of the Ca 2+‐binding B‐subunit of calcineurin (protein phosphatase‐2B) has been identified as myristic acid by fast atom bombardment mass spectrometry and gas chromatography. The sequence, myristyl‐Gly‐Asn‐Glu‐Ala‐, is very similar to that of the catalytic subunit of cyclic AMP‐dependent protein kinase, the only other protein known to contain this fatty acid. This finding, and the elution of all myristyl peptides at 57% acetonitrile on reverse phase HPLC, may facilitate the identification of other proteins with this blocking group.

Renaming the DSCR1/Adapt78 gene family as RCAN: regulators of calcineurin.

Kelvin J. A. Davies,* Gennady Ermak,* Beverley A. Rothermel, Melanie Pritchard, Joseph Heitman, Joohong Ahnn, Flavio Henrique-Silva, Dana Crawford, Silvia Canaider,** Pierluigi Strippoli,** Paolo Carinci,** Kyung-Tai Min, Deborah S. Fox, Kyle W. Cunningham, Rhonda Bassel-Duby, Eric N. Olson, Zhuohua Zhang, R. Sanders Williams, Hans-Peter Gerber,*** Merce Perez-Riba, Hisao Seo, Xia Cao, Claude B. Klee, Juan Miguel Redondo, Lois J. Maltais, Elspeth A. Bruford, Sue Povey, Jeffery D. Molkentin,**** Frank D. McKeon, Elia J. Duh, Gerald R. Crabtree,§§§§ Martha S. Cyert, Susana de la Luna, and Xavier Estivill

Concerted regulation of protein phosphorylation and dephosphorylation by calmodulin

The multiple functions of calmodulin in brain bring to light an apparent paradox in the mechanism of action of this multifunctional regulatory protein: How can the simultaneous calmodulin stimulation of enzymes with opposing functions such as cyclic nucleotide phosphodiesterases and adenylate cyclase, which are responsible for the degradation and synthesis of cAMP, respectively, be physiologically significant? The same question applies to the simultaneous activation of protein kinases (in particular calmodulin kinase II) and a protein phosphatase (calcineurin). One could propose that the protein kinase(s) and the phosphatase may be located in different cells or in different cellular compartments, and are therefore not antagonizing each other. The same result could be achieved if the specific substrates of these enzymes have different cellular localizations. This does not seem to be the case. In many areas of the brain the two enzymes and their substrates coexist in the same cell. For example, the hippocampus is rich in calmodulin kinase II, calcineruin and substrates for the two enzymes. A more general scheme is presented here, based on different mechanisms of the calmodulin regulation of the two classes of enzyme, which helps to solve this apparent inconsistency in the mechanism of action of calmodulin.

[22] Isolation and characterization of bovine brain calcineurin: A calmodulin-stimulated protein phosphatase

Publisher Summary This chapter focuses on the isolation and characterization of the bovine brain calcineurin. Calcineurin is a heterodimer composed of two subunits, a 19,000 Mr Ca2+-binding subunit, calcineurin B, and a 61,000 Mr subunit, calcineurin A, which interacts with calmodulin. A Ca2+-dependent, calmodulin-stimulated protein phosphatase activity has been found to be associated with calcineurin. The protein can be purified on the basis of its ability to interact with calmodulin after sodium dodecyl sulphate- (SDS)-gel electrophoresis, as well as by its protein phosphatase activity. Upon SDS-polyacrylamide gel electrophoresis, the small subunit of calcineurin, calcineurin B, can be readily identified as a small polypeptide with an apparent Mr between 16,000 and 15,000 that exhibits a characteristic increase in mobility in the presence of Ca2+. Once identified, this subunit can be quantitated by densitometric analysis of slab gels stained with Coomassie Brilliant Blue. The 61,000 Mr subunit, calcineurin A, is more difficult to identify on Coomassie Blue-stained gels of crude protein mixtures because of the presence of many polypeptides of similar molecular weight. However, calcineurin A, the calmodulin-binding subunit of calcineurin, can interact with calmodulin even after SDS-gel electrophoresis and it can be identified and quantitated by a modification of the [125I]calmodulin gel overlay technique.