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The plasma membrane and autoinhibited Ca 2+ -ATPases contribute to the Ca 2+ homeostasis in a wide variety of organisms. The enzymatic activity of these pumps is stimulated by calmodulin, which interacts with the target protein through the calmodulin-binding domain (CaMBD). Most information about this region is related to all calmodulin modulated proteins, which indicates general chemical properties and there is no established relation between Ca 2+ pump sequences and taxonomic classification. Thus, the aim of this study was to perform an in silico analysis of the CaMBD from several Ca 2+ -ATPases, in order to determine their diversity and to detect specific patterns and amino acid selection in different species. Patterns related to potential and confirmed CaMBD were detected using sequences retrieved from the literature. The occurrence of these patterns was determined across 120 sequences from 17 taxonomical classes, which were analyzed by a phylogenetic tree to establish phylogenetic groups. Predicted physicochemical characteristics including hydropathy and net charge were calculated for each group of sequences. 22 Ca 2+ -ATPases sequences from animals, unicellular eukaryotes, and plants were retrieved from bioinformatic databases. These sequences allow us to establish the Patterns 1(GQILWVRGLTRLQTQ), 3(KNPSLEALQRW), and 4(SRWRRLQAEHVKK), which are present at the beginning of putative CaMBD of metazoan, parasites, and land plants. A pattern 2 (IRVVNAFR) was consistently found at the end of most analyzed sequences. The amino acid preference in the CaMBDs changed depending on the phylogenetic groups, with predominance of several aliphatic and charged residues, to confer amphiphilic properties. The results here displayed show a conserved mechanism to contribute to the Ca 2+ homeostasis across evolution and may help to detect putative CaMBDs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The plasma membrane Ca 2+ -ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC 50 s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca 2+ , as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca 2+ /CaM complex. Moreover, when the peptide was incubated with CaM and Ca 2+ , a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W 1039 and F 1056 , strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L 1042 and W 1060 residues might also participate as anchors to form a 1-4-18-22 motif., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca 2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca 2+ homeostasis. Therefore, the plasma membrane Ca 2+ -ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca 2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca(2+), but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca(2+). This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca(2+), absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site ((645)R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q(660)) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells., (© 2015 Authors; published by Portland Press Limited.)

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro.

We have cloned and expressed calmodulin (CaM) from Trypanosoma cruzi, for the first time, to obtain large amounts of protein. CaM is a very well conserved protein throughout evolution, sharing 100% amino acid sequence identity between different vertebrates and 99% between trypanosomatids. However, there is 89% amino acid sequence identity between T. cruzi and vertebrate CaMs. The results demonstrate significant differences between calmodulin from T. cruzi and mammals. First, a polyclonal antibody developed in an egg-yolk system to the T. cruzi CaM recognizes the autologous CaM but not the CaM from rat. Second, it undergoes a larger increase in the alpha-helix content upon binding with Ca(2+), when compared to CaM from vertebrates. Finally, two classic CaM antagonists, calmidazolium and trifluoperazine, capable of inhibiting the action of CaM in mammals when assayed on the plasma membrane Ca(2+) pump, showed a significant loss of activity when assayed upon stimulation with the T. cruzi CaM.

Using site-directed mutagenesis, we have produced three calmodulin (CaM) mutants in which one or the two tyrosine residues of native CaM were substituted by phenylalanine. The three variants, denoted CaM(Y99F), CaM(Y138F), and CaM(Y99F/Y138F), were highly expressed in transformed Escherichia coli BL21(DE3)pLysS and purified in high yield. The three CaM mutants were able to activate the cyclic nucleotide phosphodiesterase and the plasma membrane Ca(2+)-ATPase, and present the characteristic Ca(2+)-induced electrophoretic mobility shift of native CaM. CaM(Y138F) and CaM(Y99F/Y138F), however, showed a slightly higher electrophoretic mobility than CaM(Y99F) or wild type CaM. The molar extinction coefficient of native CaM at 276 nm decreases 50% in CaM(Y99F) and CaM(Y138F), while the 276nm peak disappears in CaM(Y99F/Y138F). Terbium fluorescence studies with the different CaM mutants indicate that Y99 (but not Y138) closely interacts with Ca(2+) in the III Ca(2+)-binding domain. The epidermal growth factor receptor (EGFR) and the non-receptor tyrosine kinase c-Src phosphorylate CaM(Y99F) and CaM(Y138F) at a lesser extent than wild type CaM, while they fail to phosphorylate CaM(Y99F/Y138F) as expected. All resulting phospho-(Y)CaM species present the characteristic Ca(2+)-induced electrophoretic mobility shift observed in non-phosphorylated CaM. Quantitative analysis of the different phospho-(Y)CaM species suggests that the relative phosphorylation of Y99 and Y138 in wild type CaM by both the EGFR and c-Src is different than the respective phosphorylation of either Y99 in CaM(Y138F) or Y138 in CaM(Y99F).

Calmodulin (CaM) is phosphorylated in vitro and in vivo by multiple protein-serine/threonine and protein-tyrosine kinases. Casein kinase II and myosin light-chain kinase are two of the well established protein-serine/threonine kinases implicated in this process. On the other hand, within the protein-tyrosine kinases involved in the phosphorylation of CaM are receptors with tyrosine kinase activity, such as the insulin receptor and the epidermal growth factor receptor, and nonreceptor protein-tyrosine kinases, such as several members of the Src family kinases, Janus kinase 2, and p38Syk. The phosphorylation of CaM brings important physiological consequences for the cell as the diverse phosphocalmodulin species have differential actions as compared to nonphosphorylated CaM when acting on different CaM-dependent systems. In this review we will summarize the progress made on this topic as the first report on phosphorylation of CaM was published almost two decades ago. We will emphasize the description of the phosphorylation events mediated by the different protein kinases not only in the test tube but in intact cells, the phosphorylation-mediated changes of CaM activity, its action on CaM-dependent systems, and the functional repercussion of these phosphorylation processes in the physiology of the cell.

Calmodulin (CaM), a major intracellular Ca2+ receptor protein, has been identified and partially characterized in several trypanosomatids. The amino acid sequences of CaM from Trypanosoma cruzi and Trypanosoma brucei are known, while that from Leishmania mexicana is not. CaM from T. cruzi contains 18 amino acid substitutions, as compared with CaM from bovine brain. In addition, CaM from bovine brain contains two tyrosine residues (Tyr-99 and Tyr-138), while CaM from T. cruzi only contains Tyr-138. In the present work we show that a monoclonal antibody developed against the carboxyl-terminal region of bovine brain CaM fails to recognize CaM from both T. cruzi and L. mexicana. CaM from both parasites and from bovine brain were phosphorylated in vitro by a preparation of CaM-binding protein kinases enriched in the epidermal growth factor (EGF) receptor. Phosphoamino acids analysis demonstrated EGF-dependent phosphorylation of tyrosine residues in bovine brain CaM, while only trace amounts of tyrosine phosphorylation were detected in CaM from both trypanosomatids. These results demonstrate that the EGF receptor tyrosine kinase targets Tyr-99, but not Tyr-138, as the single major phosphorylatable residue of CaM. On the other hand, and in contrast to bovine brain CaM, there is a significant phosphorylation of serine residues in CaM from trypanosomatids which is activated by the EGF receptor via a protein-serine/threonine kinase cascade.

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] that the plasma membrane of different trypanosomatids only contains Ca(2+)-ATPase that does not show any demonstrable dependence on Mg2+, a high-affinity (Ca(2+)-Mg2+)-ATPase was demonstrated in the plasma membrane of Trypanosoma brucei. The enzyme became saturated with micromolar amounts of Ca2+, reaching a Vmax. of 3.45 +/- 0.66 nmol of ATP/min per mg of protein. The Km,app. for Ca2+ was 0.52 +/- 0.03 microM. This was decreased to 0.23 +/- 0.05 microM, and the Vmax. was increased to 6.36 +/- 0.22 nmol of ATP/min per mg of protein (about 85%), when calmodulin was present. T. brucei plasma-membrane vesicles accumulated Ca2+ on addition of ATP only when Mg2+ was present, and released it to addition of the Ca2+ ionophore A23187. In addition, this Ca2+ transport was stimulated by calmodulin. Addition of NaCl to Ca(2+)-loaded T. brucei plasma-membrane vesicles did not result in Ca2+ release, thus suggesting the absence of a Na+/Ca2+ exchanger in these parasites. Therefore the (Ca(2+)-Mg2+)-ATPase would be the only mechanism so far described that is responsible for the long-term fine tuning of the intracellular Ca2+ concentration of these parasites. The trypanocidal drug pentamidine inhibited the T. brucei plasma-membrane (Ca(2+)-Mg2+)-ATPase and Ca2+ transport at concentrations that had no effect on the Ca(2+)-ATPase activity of human or pig erythrocytes. In this latter case, pentamidine behaved as a weak calmodulin antagonist, since it inhibited the stimulation of the erythrocyte Ca(2+)-ATPase by calmodulin.

The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin. As it has been found in the Ca(2+)-ATPase from human erythrocytes, trypsin proteolysis stimulates the ATPase activity mimicking the effect of calmodulin. On the other hand, antibodies raised against the isolated Ca(2+)-ATPase from human erythrocytes are effective in recognizing the enzyme from Leishmania mexicana, thus supporting a stronger homology between both Ca(2+)-ATPases.

The intracellular Ca2+ concentration in all eukaryotic cells so far studied is 4 orders of magnitude lower than in the extracellular milieu. In trypanosomatids, the intracellular concentration of this cation is around 50 nM, even lower than in higher eukaryotics. This differential concentration is maintained by diverse transport systems at the plasma membrane level and at certain intracellular organelles. In the case of trypanosomatids it have been identified the presence of an electrophoretic uniporter at the internal membrane of the unique giant mitochondrion of these parasites, showing identical kinetics properties to the homologous system of higher eukaryotics. Thus, the low Ca2+ affinity of this system is not compatible with its putative function of maintaining intracellular Ca2+ at the submicromolar level. Contrary to previous reports, we have identified a Ca(2+)-ATPase in the plasma membrane of Leishmania braziliensis, Leishmania mexicana, Trypanosoma cruzi and Trypanosoma brucei. The enzyme possesses a high Ca2+ affinity (Km Ca2+ = 0.5 microM), is Mg(2+)-dependent and activatable by calmodulin purified from these hemoflagellates. The Ca(2+)-ATPase is sensitive to vanadate (Ki = 1 microM), thus typifying it as a "P" type ion pump. Vesicles from the plasma membrane of these parasites are able to accumulate Ca2+ against a concentration gradient. The kinetic properties of both transport and ATPase are essentially the same, thus suggesting the same molecular entity. The above results strongly suggest that the Ca(2+)-ATPase is the mechanism responsible for the long-term fine-tuning of intracellular Ca2+ at the submicromolar lever in trypanosomatids.

High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.

The Ca2(+)-ATPase of the erythrocyte plasma membrane can be activated by calmodulin, acidic phospholipids, limited proteolysis and self-association. Recently, it has been shown that different organic solvents increase both the Vmax and the Ca2+ affinity of the enzyme (Benaim, G. and De Meis, L. (1989) FEBS Lett. 244, 484-486). In this report the effects of calmodulin and dimethyl sulfoxide (20%, v/v) on the Ca2(+)-ATPase are compared. Dimethyl sulfoxide also elicits the appearance of the low-affinity binding site, which in this enzyme is strictly dependent on calmodulin. Dimethyl sulfoxide increases the Ca2+ affinity of the enzyme in a manner similar to that observed with the use of calmodulin and of acidic phospholipids. This was tested using both native and partially trypsinized ATPase. When activated by calmodulin the enzyme is inhibited by compound 48/80, trifluoperazine and calmidazolium. When activated by dimethyl sulfoxide the enzyme is still inhibited by calmidazolium but is no longer inhibited by either compound 48/80 or trifluoperazine. Activation of the ATPase promoted by either calmodulin or dimethyl sulfoxide is abolished when the Ca2+ concentration is raised from 10 microM to 2 mM. The effect of dimethyl sulfoxide is also abolished by 20 mM Pi. In the presence of 1 to 10 mM Ca2+ the ATPase catalyzes an ATP in equilibrium Pi exchange. The rate of exchange increases several fold when dimethyl sulfoxide is included in the assay medium.