Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C

The trigger Ca2+-binding sites in troponin C, those which initiate muscle contraction, are thought to be the first two of four potential sites (sites I-IV). In cardiac troponin C, the first Ca2+-binding site is inactive, and initiation of contraction in cardiac muscle appears to involve only the second site. To study this phenomenon and associated Ca2+-dependent protein conformational changes in cardiac troponin C, the cDNA for the chicken protein was incorporated into a bacterial expression plasmid to allow site-specific mutagenesis. Ca2+-binding site I was activated by deletion of Val-28 and conversion of amino acids 29-32 to those found at the first four positions in the active site I of fast skeletal troponin C. In a series of proteins, Ca2+-binding site II was inactivated by mutation of amino acids Asp-65, Asp-67, and Gly-70. All mutated proteins exhibited the predicted calcium-binding characteristics. The single mutation of converting Asp-65 to Ala was sufficient to inactivate site II. Ca2+-dependent conformational changes in the normal and mutated proteins were monitored by labeling with a sulfhydryl-specific fluorescent dye. Activation of Ca2+-binding site I or inactivation of site II, eliminated the large Ca2+-dependent increase in fluorescence seen in the wild type protein and there was, instead, a Ca2+-dependent decrease in fluorescence. All mutant proteins could associate with troponin I and troponin T to form a troponin complex. Activation of Ca2+-binding site I changed the characteristics of contraction in skinned slow skeletal muscle fibers such that the response to Ca2+ was more cooperative. Inactivation of Ca2+-binding site II abolished Ca2+-dependent contraction in skinned muscle fibers. The data provide a direct demonstration that Ca2+-binding site II in cardiac troponin C is essential for triggering muscle contraction and support the hypothesis that site I functions to modify the characteristics of contraction.     

Effect of myosin cross-bridge interaction with actin on the Ca2(+)-binding properties of troponin C in fast skeletal myofibrils

Ca2+ binding to fast skeletal muscle troponin C reincorporated into troponin C-depleted (CDTA-treated) myofibrils has been measured directly by using 45Ca and indirectly by using a fluorescent probe. Direct Ca2(+)-binding measurements have shown that the Ca2+ affinity of the low-affinity sites is enhanced in the absence of ATP and conversely reduced when myosin is selectively extracted from myofibrils, compared to the Ca2+ affinity in the presence of ATP. Fluorescence intensity changes of a dansylaziridine label at the Met-25 residue of troponin C have shown the same Ca2(+)-sensitivity whether or not ATP is present, while much lower Ca2(+)-sensitivity is seen in the myosin-extracted myofibrils. Since the Met-25 residue is in the amino terminal side alpha-helix of Ca2(+)-binding site I and far from Ca2(+)-binding site II in the primary structure, Ca2+ binding to site II has been evaluated by assuming that the fluorescence change monitors Ca2+ binding to site I alone. Ca2+ binding to site II thus estimated has shown high positive cooperativity only in the presence of ATP and has been found to be nearly proportional to the activation of myofibrillar ATPase, suggesting that Ca2(+)-binding site II is directly involved in the activation of myofibrillar ATPase activity. On the other hand, Ca2(+)-binding site I has been suggested to regulate the interaction of weakly binding cross-bridges with the thin filament, since the fluorescence change in the presence of ATP is saturated at the free Ca2+ concentration required for the activation of myofibrillar ATPase.     

Amino acid sequences of the two major isoforms of troponin C from crayfish

The primary structure of the two major isoforms (alpha and gamma) of troponin C (TnC) from crayfish tail muscle has been determined by the application of manual and automated Edman degradation procedures to fragments generated by suitable chemical and proteolytic cleavages. Both amino acid sequences commence with an acetylated methionyl residue and contain 150 amino acid residues, including a single proline residue at position 29 and 2 residues of tyrosine at positions 95 and 102. No cysteine or tryptophan are present. The molecular weights calculated for alpha- and gamma-TnC are 17,157 and 16,974, respectively. The two crayfish proteins are invariable at 129 positions and conserved at 11 others. Pairwise comparisons show that the two sequences are 33-39% identical with those of seven TnCs reported so far and 39% identical with that of bovine brain calmodulin. The N-terminal end of about 10 residues, found in vertebrate TnCs, is absent in crayfish TnCs. In the latter proteins, domains I and III appear as abortive Ca2+-binding sites due to nonconservative amino acid replacements at the key Ca2+-coordinating positions in their loops. The remaining two Ca2+-binding loops (II and IV) show a remarkable similarity with the Ca2+-specific loops (I and II) found in vertebrate TnCs. These findings are consistent with the Ca2+-binding data (Wnuk, W. (1989) J. Biol. Chem. 264, 18240-18246) which indicate the presence of two Ca2+-specific sites in crayfish TnCs. These two sites display the same affinity for Ca2+ (log KCa = 4.3) on gamma-TnC but differ in their affinity (log KCa = 6.0 and 4.1) on alpha-TnC. The only structural difference between the dodecapeptide loops II and IV in both alpha- and gamma-TnC, which correlates with the existence of the high affinity (log KCa = 6.0) Ca2+-specific site on alpha-TnC, is position 11 occupied by a methionyl residue in the loop IV of alpha-TnC as opposed to negatively charged residues found in the other three loops. This suggests that the high affinity Ca2+-specific site on alpha-TnC is located in domain IV. Since the Ca2+-binding studies show that the formation of the complex of crayfish troponin I (TnI) with alpha- and gamma-TnC increases significantly the affinity of only one of their two Ca2+-specific sites and this TnI-sensitive site is not the high affinity Ca2+-specific site on alpha-TnC, we conclude that the binding of Ca2+ to site II controls the Ca2+-dependent interaction between crayfish TnCs and TnI.     

A series of point mutations reveal interactions between the calcium-binding sites of calmodulin.

Calmodulin is a member of the "EF-hand" family of Ca(2+)-binding proteins. It consists of two homologous globular domains, each containing two helix-loop-helix Ca(2+)-binding sites. To examine the contribution of individual Ca(2+)-binding sites to the Ca(2+)-binding properties of CaM, a series of four site-directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca(2+)-binding loops has been changed to a glutamine. One-dimensional 1H-NMR has been used to monitor Ca(2+)-induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild-type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca(2+)-binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF-hand Ca(2+)-binding loops greatly decreases the Ca(2+)-binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca(2+)-binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca(2+)-binding affinities of the partner C-terminal site. In both the N- and C-terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C-terminus, the conformational consequences of mutating site III or site IV are strikingly different.     

Isolation of S-100 binding proteins from brain by affinity chromatography

S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions.     

Theoretical estimation of the calcium-binding constants for proteins from the troponin C superfamily based on a secondary structure prediction method. II. Applications

Ca2+-binding properties of the following proteins, classified as members of the troponin C (TNC) superfamily have been discussed: TNCs, calmodulins (CaMs), vitamin D-dependent calcium-binding proteins (CaBPs), myosin light chains (LCs), S-100 chains, parvalbumins (PVs), oncomodulin (OCM), sarcoplasmic calcium binding proteins (SCPs), calcineurin B (CB) and calcium vector protein (CaVP). Assuming the most probable domain pairing, the Ca2+-binding constants of these proteins have been predicted from their sequences using the method presented in the preceding paper. The results are critically compared with the available experimental data. For some proteins (TNCs, CaMs, CaBPs, LCs, CB and CaVP) our predictions are consistent with the experimental results. For the others, substantial discrepancies between the predicted and measured KCa values are observed. They result from some structural peculiarities of those proteins: a unique, three-domain organization in the case of PVs and OCM, unusual sequences of binding loops in the case of S-100 and a lack of a standard helix-loop-helix organization of Ca2+-binding domains in the case of SCPs.     

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.     

Measurement of Ca2+-binding constants of proteins and presentation of the CaLigator software

The complexity of Ca2+ cell signaling is dependent on a plethoria of Ca2+-binding proteins that respond to signals in different ranges of Ca2+ concentrations. Since the function of these proteins is directly coupled to their Ca2+-binding properties, there is a need for accurately determined equilibrium Ca2+-binding constants. In this work we outline the experimental techniques available to determine Ca2+-binding constants in proteins, derive the models used to describe the binding, and present CaLigator, software for least-square fitting directly to the measured quantity. The use of the software is illustrated for Ca2+-binding data obtained for two deamidated forms of calbindin D(9k), either an isospartate-56 (beta form) or a normal Asp-56 (alpha form). Here, the Ca2+-binding properties of the two isoforms have been studied using the chelator method. The alpha form shows similar Ca2+-binding properties to the wild type while the beta form has lost both cooperativety and affinity.     

The Ca2+/calmodulin system in neuronal hyperexcitability

Calmodulin (CaM) is a major Ca2+-binding protein in the brain, where it plays an important role in the neuronal response to changes in the intracellular Ca2+ concentration. Calmodulin modulates numerous Ca2+-dependent enzymes and participates in relevant cellular functions. Among the different CaM-binding proteins, the Ca2+/CaM dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. Therefore, the role of the Ca2+/CaM signalling system in different neurotoxicological or neuropathological conditions associated to alterations in the intracellular Ca2+ concentration is a subject of interest. We here report different evidences showing the involvement of CaM and the CaM-binding proteins above mentioned in situations of neuronal hyperexcitability induced by convulsant agents. Signal transduction pathways mediated by specific CaM binding proteins warrant future study as potential targets in the development of new drugs to inhibit convulsant responses or to prevent or attenuate the alterations in neuronal function associated to the deleterious increases in the intracellular Ca2+ levels described in different pathological situations.     

Three Ca2+-binding proteins from porcine liver and intestine differ immunologically and physicochemically and are distinct in Ca2+ affinities

Intestinal brush-border-derived membrane vesicles contain, after demembranation in the presence of Ca2+, a subset of polypeptides that are specifically solubilized by the addition of Ca2+ chelators. As described previously, this fractionation scheme leads to the enrichment of two major proteins (I and II), one of which has been shown to be identical to the cellular p36K target of Rous sarcoma virus-encoded tyrosine-specific protein kinase (Gerke, V., and Weber, K., (1984) EMBO J. 3, 227-233). We have applied a similar protocol to membrane vesicles from porcine liver and purified a third Ca2+-binding protein (III). All three proteins had wide tissue distributions, and were absent from brain, red blood cells, and cardiac and skeletal muscle. Relative amounts varied between tissues, with protein I low in liver and protein III very low in intestine. Despite their similar extractability the three proteins (I, II, and III) are clearly distinct as far as immunological, biochemical, and physicochemical properties are concerned. They also show characteristic differences in their affinities for Ca2+ ions. The association constants of Ca2+ binding for proteins I and III have been estimated by means of indirect methods to be 10(4) M-1 (protein I) and 10(6) M-1 (protein III), while the direct Hummel-Dreyer method reveals Ca2+ binding to protein II, characterized by an association constant of 0.4 X 10(5) M-1 in the absence and 0.2 X 10(5) M-1 in the presence of 2 mM MgCl2. Conformational changes upon binding Ca2+ are described for protein II using circular dichroism, fluorescence emission, and UV difference spectra. These alterations could be attributed to an increased exposure of tyrosine and tryptophan residues to a more aqueous environment, and led to increased hydrophobicity of protein II that would explain the observed Ca2+-dependent interaction with hydrophobic matrices like phenyl-Sepharose.     

Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers

In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.     

Intralumenal sarcoplasmic reticulum Ca(2+)-binding proteins

The sarcoplasmic reticulum (SR) controls the level of intracellular Ca2+ in cardiac and skeletal muscle by storing and releasing Ca2+. A set of intralumenal SR Ca(2+)-binding proteins has been identified that may serve important roles in SR Ca2+ storage and mobilization. The most prominent of these SR proteins, calsequestrin, is discretely localized to junctional SR. Other intralumenal proteins are more widely distributed throughout the SR. All of these intralumenal SR Ca(2+)-binding proteins are acidic, stain blue with dye Stains-All, and appear to be substrates for casein kinase II. The biochemistry and cell biology of lumenal SR proteins may conform to a paradigm now emerging from the study of endoplasmic reticulum proteins.     

Ca2+-binding proteins from bovine brain including a potent inhibitor of protein kinase C.

We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.     

Effects of modifying individual amino or carboxyl groups on the affinity of calmodulin for calcineurin

The effects of modifying individual lysyl, aspartyl, or glutamyl residues in calmodulin on its ability to bind to the neural phosphatase calcineurin have been investigated using a competitive binding method termed "label selection." Samples of calmodulin were radiochemically labeled at a low level (0.03-0.6 group/molecule) by acetylation of amino groups or coupling carboxyl groups with ethanolamine to produce preparations containing predominantly single-site modified and unmodified molecules. These preparations were incubated in a 5-10-fold molar excess with bovine calcineurin under conditions appropriate for complex formation. The bound population was isolated, and the level of modification of each reactive residue was compared with the level in the corresponding group in the intial unselected preparation to determine if molecules modified at specific sites had been selected for or against during the competition for complex formation. Significant selection was observed against molecules modified at Lys21, Asp64, Glu67, Lys75, Glu84, Glu114, Asp118, or Lys148, whereas modification of Glu83 increased binding. The modification of other groups, including components of the four Ca2+-binding sites, had no effect on the interaction. Glu67, a Ca2+-liganding residue in Ca2+-binding site II that may regulate the orientation of this site in relation to the central helix, had the strongest influence on complex formation. Most of the residues identified form a nearly linear array in the three-dimensional structure of calmodulin and indicate the location of an extended surface for interaction with calcineurin and other enzymes.     

Structures of Anabaena calcium-binding protein CcbP: insights into Ca2+ signaling during heterocyst differentiation

Ca2+-binding proteins play pivotal roles in both eukaryotic and prokaryotic cells. CcbP from cyanobacterium Anabaena sp. strain PCC 7120 is a major Ca2+-binding protein involved in heterocyst differentiation, a process that forms specialized nitrogen-fixing cells. The three-dimensional structures of both Ca2+-free and Ca2+-bound forms of CcbP are essential for elucidating the Ca2+-signaling mechanism. However, CcbP shares low sequence identity with proteins of known structures, and its Ca2+-binding sites remain unknown. Here, we report the solution structures of CcbP in both Ca2+-free and Ca2+-bound forms determined by nuclear magnetic resonance spectroscopy. CcbP adopts an overall new fold and contains two Ca2+-binding sites with distinct Ca2+-binding abilities. Mutation of Asp38 at the stronger Ca2+-binding site of CcbP abolished its ability to regulate heterocyst formation in vivo. Surprisingly, the β-barrel subdomain of CcbP, which does not participate in Ca2+-binding, topologically resembles the Src homology 3 (SH3) domain and might act as a protein-protein interaction module. Our results provide the structural basis of the unique Ca2+ signaling mechanism during heterocyst differentiation.     

The primary structure of a new Mr 18,000 calcium vector protein from amphioxus

The amino acid sequence of a new Ca2+-binding protein (CaVP) from Amphioxus muscle (Cox, J. A., J. Biol. Chem. 261, 13173-13178) has been determined. The protein contains 161 amino acid residues and has a molecular weight of 18,267. The N terminus is blocked by an acetyl group. The two functional Ca2+-binding sites have been localized based on homology with known Ca2+-binding domains, on internal homology and on secondary structure prediction, and appear to be the domains III and IV. The C-terminal half of CaVP, which contains the two Ca2+-binding sites, shows a remarkable similarity with human brain calmodulin (45%) and with rabbit skeletal troponin C (40%). Functional domain III contains 2 epsilon-N-trimethyllysine residues in the alpha-helices flanking the Ca2+-binding loop. Sequence determination revealed two abortive Ca2+-binding domains in the N-terminal half of CaVP with a similarity of 24 and 30% as compared with calmodulin and troponin C, respectively. This half is also characterized by the presence of a disulfide bridge linking the N-terminal helix of domain I to the C-terminal helix of domain II. This disulfide bond is very resistant to reduction in the native state, but not in denatured CaVP. The optically interesting aromatic chromophores (2 tryptophan and 1 tyrosine residues) are all located in the nonfunctional domain II.     

Sodium-23 and lanthanum-139 nuclear magnetic resonance studies of cation binding to aqualysin I,a thermostable serine protease

Aqualysin I has at least two Ca2+-binding sites that have different affinities for Ca2+. The binding of various metal ions to aqualysin I was studied using 23Na- and 139La-NMR spectrometry. Evidence is presented that Ca2+, La3+, and Na+ bind to the low-affinity Ca2+-binding site of aqualysin I, but Mg2+ does not. Our results confirm that binding of metals at the low-affinity Ca2+-binding site is essential for thermostabilization, since the addition of Mg2+ did not result in thermostabilization. La3+ was found to bind to both the low-affinity Ca2+-binding site and an additional metal ion-binding site that can also be involved in the thermostabilization of aqualysin I.     

Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases

Ca2+ ATPases deplete the cytosol of Ca2+ ions and are crucial to cellular Ca2+ homeostasis. The PMC1 gene of Saccharomyces cerevisiae encodes a vacuole membrane protein that is 40% identical to the plasma membrane Ca2+ ATPases (PMCAs) of mammalian cells. Mutants lacking PMC1 grow well in standard media, but sequester Ca2+ into the vacuole at 20% of the wild-type levels. pmc1 null mutants fail to grow in media containing high levels of Ca2+, suggesting a role of PMC1 in Ca2+ tolerance. The growth inhibitory effect of added Ca2+ requires activation of calcineurin, a Ca2+ and calmodulin-dependent protein phosphatase. Mutations in calcineurin A or B subunits or the inhibitory compounds FK506 and cyclosporin A restore growth of pmc1 mutants in high Ca2+ media. Also, growth is restored by recessive mutations that inactivate the high-affinity Ca(2+)-binding sites in calmodulin. This mutant calmodulin has apparently lost the ability to activate calcineurin in vivo. These results suggest that activation of calcineurin by Ca2+ and calmodulin can negatively affect yeast growth. A second Ca2+ ATPase homolog encoded by the PMR1 gene acts together with PMC1 to prevent lethal activation of calcineurin even in standard (low Ca2+) conditions. We propose that these Ca2+ ATPase homologs are essential in yeast to deplete the cytosol of Ca2+ ions which, at elevated concentrations, inhibits yeast growth through inappropriate activation of calcineurin.     

Mammalian brain phosphoproteins as substrates for calcineurin

Calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase found in several tissues, is highly concentrated in mammalian brain. In an attempt to identify endogenous brain substrates for calcineurin, kinetic analyses of the dephosphorylation of several well-characterized phosphoproteins purified from brain were performed. The proteins studied were: G-substrate, a substrate for cyclic GMP-dependent protein kinase; DARPP-32, a substrate for cyclic AMP-dependent protein kinase; Protein K.-F., a substrate for a cyclic nucleotide- and Ca2+-independent protein kinase; and synapsin I, a substrate for cyclic AMP-dependent (site I) and a Ca2+/calmodulin-dependent protein kinase (site II). Calcineurin dephosphorylated each of these proteins in a Ca2+/calmodulin-dependent manner. Similar Km values were obtained for each substrate: G-substrate, 3.8 microM; DARPP-32, 1.6 microM; Protein K.-F., approximately 3 microM (S0.5); synapsin I (site I), 7.0 microM; synapsin I (site II), 4.4 microM. However, significant differences were obtained for the maximal rates of dephosphorylation. The kcat values were: G-substrate, 0.41 s-1; DARPP-32, 0.20 s-1; Protein K.-F., 0.7 s-1; synapsin I (site I), 0.053 s-1; synapsin I (site II), 0.040 s-1. Comparisons of the catalytic efficiency (kcat/Km) for each substrate indicated that DARPP-32, G-substrate, and Protein K.-F. are all potential substrates for calcineurin in vivo.     

S100-annexin complexes--biology of conditional association

S100 proteins and annexins both constitute groups of Ca2+-binding proteins, each of which comprises more than 10 members. S100 proteins are small, dimeric, EF-hand-type Ca2+-binding proteins that exert both intracellular and extracellular functions. Within the cells, S100 proteins regulate various reactions, including phosphorylation, in response to changes in the intracellular Ca2+ concentration. Although S100 proteins are known to be associated with many diseases, exact pathological contributions have not been proven in detail. Annexins are non-EF-hand-type Ca2+-binding proteins that exhibit Ca2+-dependent binding to phospholipids and membranes in various tissues. Annexins bring different membranes into proximity and assist them to fuse, and therefore are believed to play a role in membrane trafficking and organization. Several S100 proteins and annexins are known to interact with each other in either a Ca2+-dependent or Ca2+-independent manner, and form complexes that exhibit biological activities. This review focuses on the interaction between S100 proteins and annexins, and the possible biological roles of these complexes. Recent studies have shown that S100-annexin complexes have a role in the differentiation of gonad cells and neurological disorders, such as depression. These complexes regulate the organization of membranes and vesicles, and thereby may participate in the appropriate disposition of membrane-associated proteins, including ion channels and/or receptors.