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Biomedical subjects

E Carafoli

Publications and source records attributed to E Carafoli.

At least 37 records · Page 2Linked to original sources
PubMed1999

Serine/threonine phosphorylation of calmodulin modulates its interaction with the binding domains of target enzymes.

The interaction of serine/threonine-phosphorylated calmodulin with synthetic peptides corresponding to the calmodulin-binding domains of six enzymes has been studied by fluorescence spectroscopy. For five peptides, the dissociation constant of the calmodulin-peptide complex (K(d)) increased when calmodulin was phosphorylated. An increase of more than one order of magnitude was observed with peptides derived from smooth-muscle myosin light-chain kinase and cAMP phosphodiesterase. In contrast, only a slight increase in K(d) was noted with two peptides derived from the plasma membrane Ca(2+)-ATPase and for the peptide derived from nitric oxide synthase. No significant change in affinity was detected with the peptide derived from calcineurin. In contrast, a decrease in the dissociation constant was observed with the peptide derived from the Ca(2+)-calmodulin dependent kinase II. Phosphorylation also affected the peptide-calmodulin binding stoichiometry: a decrease from two to one binding sites was observed with the peptides derived from myosin light-chain kinase and phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Microinjection of Ca2+ store-enriched microsome fractions to dividing newt eggs induces extra-cleavage furrows via inositol 1,4,5-trisphosphate-induced Ca2+ release.

The cleavage signal transferred to the future cleavage cortex during anaphase has been proposed as "cleavage stimulus," but no signal has proved to induce cleavage furrows. The local Ca2+ transient along the cleavage furrow has been reported, but the Ca2+ source has remained unknown. To address these questions, we studied functions of Ca2+ stores in dividing newt eggs and found that microinjection of the Ca2+ store-enriched microsome fraction to the dividing newt egg induced a local extra-cleavage furrow at the injection site in 64-67% of the injected newt eggs while coinjection with inositol 1,4, 5-trisphosphate receptor (IP(3)R) antagonists heparin or anti-type 1-IP(3)R antibody clearly suppressed this induction (5 and 11% in induction rates, respectively). Injection of cerebellar microsomes from the type 1-IP(3)R-deficient mice induced extracleavage furrows albeit at a low rate (19%). Our observations strongly suggest that Ca2+ stores with IP(3)R induce and position a cleavage furrow via IP(3)-induced Ca2+ release (IICR) as Ca(2+)-releasing machinery and putative cleavage stimulus itself.

Anaphase↗
PubMed1999

NMR solution structure of a complex of calmodulin with a binding peptide of the Ca2+ pump.

The three-dimensional structure of the complex between calmodulin (CaM) and a peptide corresponding to the N-terminal portion of the CaM-binding domain of the plasma membrane calcium pump, the peptide C20W, has been solved by heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy. The structure calculation is based on a total of 1808 intramolecular NOEs and 49 intermolecular NOEs between the peptide C20W and calmodulin from heteronuclear-filtered NOESY spectra and a half-filtered experiment, respectively. Chemical shift differences between free Ca(2+)-saturated CaM and its complex with C20W as well as the structure calculation reveal that C20W binds solely to the C-terminal half of CaM. In addition, comparison of the methyl resonances of the nine assigned methionine residues of free Ca(2+)-saturated CaM with those of the CaM/C20W complex revealed a significant difference between the N-terminal and the C-terminal domain; i.e., resonances in the N-terminal domain of the complex were much more similar to those reported for free CaM in contrast to those in the C-terminal half which were significantly different not only from the resonances of free CaM but also from those reported for the CaM/M13 complex. As a consequence, the global structure of the CaM/C20W complex is unusual, i.e., different from other peptide calmodulin complexes, since we find no indication for a collapsed structure. The fine modulation in the peptide protein interface shows a number of differences to the CaM/M13 complex studied by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638]. The unusual binding mode to only the C-terminal half of CaM is in agreement with the biochemical observation that the calcium pump can be activated by the C-terminal half of CaM alone [Guerini, D., Krebs, J., and Carafoli, E. (1984) J. Biol. Chem. 259, 15172-15177].

Amino Acid Sequence↗
PubMed1999

Calcineurin controls inositol 1,4,5-trisphosphate type 1 receptor expression in neurons.

In the central nervous system, release of Ca2+ from intracellular stores contributes to numerous functions, including neurotransmitter release and long-term potentiation and depression. We have investigated the developmental profile and the regulation of inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) in primary cultures of cerebellar granule cells. The expression of both receptor types increases during development. Whereas the expression of type 1 IP3R appears to be regulated by Ca2+ influx through L type channels or N-methyl-D-aspartate (NMDA) receptors, RyR levels increase independently of Ca2+. The main target of Ca2+-influx-regulating IP3R expression is the Ca2+ calmodulin-dependent protein phosphatase calcineurin, because pharmacological blockade of this protein abolishes IP3R expression. Although calcineurin has been shown to regulate the phosphorylation state of the IP3R, the effect described here is at the transcriptional level because IP3R mRNA changes in parallel with protein levels. Thus, calcineurin plays a dual role in IP3R-mediated Ca2+ signaling: it regulates IP3R function by dephosphorylation in the short-term time scale and IP3R expression over more extended periods.

Animals↗
PubMed1999

Identification and functional expression of the plasma membrane calcium ATPase gene family from Caenorhabditis elegans.

Calcium-pumping ATPases are an essential component of the intracellular calcium homeostasis system and have been characterized in a large variety of species and cell types. In mammalian genomes, these proteins are encoded by gene families whose individual members feature complex tissue-specific expression and alternative splicing. In the search for a less complex system that is more amenable to genetic manipulation, we have identified a family of three genes (mca-1, mca-2, and mca-3) encoding putative calcium ATPases in the Caenorhabditis elegans Genome Project data and completed their transcript structure. In this work, we report the cloning and functional expression of the mca-1 gene, which encodes a calcium-stimulated ATPase whose features resemble those of the plasma membrane calcium adenosine triphosphatase family of mammalian cells and appears to be regulated by a multipartite promoter.

Amino Acid Sequence↗
PubMed1999

The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+.

Plasma membrane Ca2+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1-3 days after plating, but they became highly expressed after 7-9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca2+ channels, significantly increasing cytosolic Ca2+. Changes in the concentration of Ca2+ in the culturing medium affected the expression of the pumps. L-type Ca2+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca2+ through the activation of glutamate-operated Ca2+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/endoplasmic reticulum calcium pump was induced by the depolarizing conditions.

Animals↗
PubMed1999

Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Expression, partial purification and functional properties of themuscle-specific calpain isoform p94.

The muscle-specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His-tagged p94 bound calcium with high affinity, and their autolytic activity required Ca2+. The sensitivity of the catalytic activity of the recombinant protease to Ca2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m- and micro-calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle-specific calpain isoform.

Animals↗
PubMed1999

Plasma membrane calcium ATPase isoforms in astrocytes.

The plasma membrane Ca(2+)-ATPase (PMCA) is an essential component of the machinery responsible for cellular Ca(2+) homeostasis. Together with the Na(+)/Ca(2+) exchanger, the plasma membrane Ca(2+)-ATPase (PMCA) is responsible for the extrusion of Ca(2+) from the cytosol. Although both PMCAs and Na(+)/Ca(2+) exchangers are present in high amounts in the brain, it is thought that only the latter localize to glia. This study investigates whether PMCAs are also present in astrocytes and thus are components of Ca(2+) signalling in this cell type. Membrane proteins and mRNA were isolated from primary cultures of rat cortical astrocytes and C6 glioma cells. PMCA isoforms were investigated with isoform specific antibodies and the splice variant pattern was studied in RT-PCR experiments using specific oligonucleotides. The PMCA1, 2, and 4 isoforms were detected in rat cortical astrocytes, whereas only PMCA1 and 2 were found in C6 cells. While neurons express both the CI and CII splice variants, only the splice variant CI of PMCA1, 2, and 4 was detected in astrocytes. Thus, the PMCA pump is present in mammalian glial cells. These results also show that the amounts of PMCA1 and 4 isoforms in astrocytes are comparable to those found in neurons. In contrast, astrocytes contain smaller amounts of PMCA2. Furthermore, PMCA2 and PMCA4 underwent an evident time dependent up-regulation in astrocytes cultured in vitro.

Animals↗
PubMed1998

The effect of ethanol on the plasma membrane calcium pump is isoform-specific.

The effect of ethanol has been studied on four different isoforms of the plasma membrane Ca2+-ATPase expressed in Sf9 cells with the help of the baculovirus system. The PMCA2CI protein was maximally activated by 0.5% ethanol, a concentration 8-10 times lower than that needed to obtain the same effect on the PMCA4 protein or on the pump of erythrocyte membranes, which is a mixture of isoforms 1 and 4. Experiments performed with truncated pumps indicated that the stimulation by ethanol was lost if the C-terminal region between Lys1065 and Lys1161, encompassing the calmodulin binding domain, was removed. These observations indicate that the stimulation is the result of a direct interaction of ethanol with the C-terminal regulatory domain of the Ca2+ pump.

Animals↗
PubMed1998

A novel molecular determinant for cAMP-dependent regulation of the frog heart Na+-Ca2+ exchanger.

Na+-Ca2+ exchanger is one of the major sarcolemmal Ca2+ transporters of cardiac myocytes. In frog ventricular myocytes the exchanger is regulated by isoproterenol via a beta-adrenoreceptor/adenylate-cyclase/cAMPdependent signaling pathway providing a molecular mechanism for the relaxant effect of the hormone. Here, we report on the presence of a novel exon of 27-base pair insertion, which generates a nucleotide binding motif (P-loop) in the frog cardiac Na+-Ca2+ exchanger. To examine the functional role of this motif, we constructed a full-length frog heart Na+-Ca2+ exchanger cDNA (fNCX1a) containing this exon. The functional expression of fNCX1a in oocytes showed characteristic voltage dependence, divalent (Ni2+, Cd2+) inhibition, and sensitivity to cAMP in a manner similar to that of native exchanger in frog myocytes. In oocytes expressing the dog heart NCX1 or the frog mutant (DeltafNCX1a) lacking the 9-amino acid exon, cAMP failed to regulate Na+-dependent Ca2+ uptake. We suggest that this motif is responsible for the observed cAMP-dependent functional differences between the frog and the mammalian hearts.

Adenylyl Cyclases↗
PubMed1998

Phosphorylation of calmodulin alters its potency as an activator of target enzymes.

Previous work has shown that calmodulin (CaM) is constitutively phosphorylated in rat liver, probably by casein kinase II [Quadroni, M., James, P., and Carafoli, E. (1994) J. Biol. Chem. 269, 16116-16122]. A procedure is now described for the isolation of the phosphorylated forms of calmodulin (PCaM) free from CaM, since in vitro phosphorylation experiments yield a 50:50 mixture of 3-4 times phosphorylated CaM and native CaM. The activation of six target enzymes by PCaM was tested: myosin light chain kinase, 3',5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, Ca2+-CaM-dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase, and CaM-kinase II. In general, the phosphorylation of CaM caused a decrease in enzyme binding affinity, increasing the Kact by 2-4-fold for MLCK, PDE, PM Ca2+-ATPase, and calcineurin. The Vmax at saturating concentrations of PCaM was less affected, with the exception of CaM-kinase II, which was only minimally activated by PCaM and NOS whose Vmax was increased 2.6 times by PCaM with respect to CaM. Phosphorylation of calmodulin had very little effect on the binding of calcium to the enzyme despite the fact that Ser 101 which is phosphorylated is located in the third calcium binding loop. CD measurements performed on CaM and PCaM indicated that phosphorylation causes a marked decrease in the alpha-helical content of the protein. Phosphorylated CaM is very prone to dephosphorylation and was thus tested as a substrate for several phosphatases. It was unaffected by calcineurin (PP2B), but was a reasonable substrate for the pleiotropic phosphatases PP1gamma and PP2A.

Animals↗
PubMed1998

Cloning of the multipartite promoter of the sodium-calcium exchanger gene NCX1 and characterization of its activity in vascular smooth muscle cells.

The sodium-calcium exchange activity is mediated by proteins encoded in a small gene family, of which the gene NCX1 is ubiquitously expressed in mammalian tissues. In this study, the multipartite promoter of this gene was analyzed in the human and rat genomes by means of DNA cloning, reverse transcriptase-polymerase chain reaction, and transient transfection of fusion constructs with the firefly luciferase gene into cultured rat aortic smooth muscle cells. The gene-proximal promoter, located 30 kilobase pairs (kb) away from the first coding exon 2, has features of a GC-rich housekeeping promoter and is apparently always active; in specific tissues, however, it is augmented by one or two additional promoters, located either within 1.5 kb upstream of it, or 35 kb upstream. The gene proximal promoter shows the highest activity in aortic smooth muscle cells. In mammalian species transcripts from all three promoters undergo splicing via an intermediate, containing two noncoding exons, of which the downstream one is normally not present in the terminal splicing product.

Alternative Splicing↗
PubMed1998

The calcium pump of the plasma membrane: membrane targeting, calcium binding sites, tissue-specific isoform expression.

The two Ca2+ pumps of higher eucaryotes are strictly targeted to different membrane systems: the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). Chimeric constructs of the two pumps expressed in COS-7 cells have revealed a strong signal for endoplasmic reticulum retention in the N-terminal cytosolic portion of the SERCA pump: the signal is contained in a stretch of 28 amino acids that follows the N-terminus. A second, but masked, endoplasmic reticulum retention signal is contained in a cytosolic C-terminal sequence immediately preceding the calmodulin-binding domain of the Ca2+ pump. Selective mutations on the SERCA pump have led to the conclusion that 5 conserved residue membrane domains (TM)4, 5, and 6 form the Ca2+ channel through the pump protein. A comparative sequence inspection has failed to reveal any of these residues in TM5 of the PMCA pump. Mutation of the conserved residue in TM4 and of two in TM6 abolished the ability of the pump to form the Ca(2+)-dependent phosphoenzyme. However, one of the mutations (N979, TM6) also caused retention of the PMCA pump in the reticulum, suggesting structural alterations. Of the four basic isoforms of the pump, two (1, 4) are ubiquitously expressed, two (2, 3) are essentially brain specific. Isoform 2 has the highest calmodulin affinity. Primary cultures of cerebellar granule cells from newborn rats did not express isoforms 2 and 3 at plating time. Incubation of the cells in depolarizing concentrations of KCl, which promote Ca2+ influx, promoted the expression of isoforms 2 and 3, and of a brain specific spliced variant of isoform 1. Incubation of the cells in L-type Ca2+ channel blockers abolished the upregulation of the pump genes.

Amino Acid Sequence↗
PubMed1997

Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit.

The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.

Animals↗
PubMed1997

Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli.

Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST). GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.

Animals↗