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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.)

The plasma-membrane Ca(2+)-ATPase is a key enzyme in the regulation of the intracellular Ca(2+) concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca(2+). In view of the fact that the Ca(2+)-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca(2+)-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca(2+) and the V(max) of the enzyme. Furthermore, this second messenger also stimulates Ca(2+) transport in inside-out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca(2+)-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca(2+)-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca(2+) concentration.

Cytoplasmic pH (pHi) regulation was studied in Trypanosoma cruzi epimastigotes using fluorescent probes. Steady-state pHi was maintained even in the absence of extracellular Na+ or K+, but was significantly decreased in the absence of Cl-. Acid-loaded epimastigotes regained normal pHi by a process that was ATP-dependent and sensitive to N-ethylmaleimide, dicyclohexyl-carbodi-imide and diethylstiboestrol, suggesting involvement of a H(+)-pumping ATPase. Recovery from an acid load was independent of extracellular Na+ or K+ and insensitive to omeprazole, vanadate and low concentrations of bafilomycin A1. Using the fluorescent probe bisoxonol to measure the membrane potential of intact cells, acid loading of epimastigotes was shown to result in a dicyclohexylcarbodi-imide-sensitive hyperpolarization, which suggests electrogenic pumping of protons across the plasma membrane. Addition of glucose, but not of 6-deoxyglucose, produced a transient cellular acidification of possible metabolic origin, and increased the rate of recovery from an acid load. Taken together, these results are consistent with an important role of a H(+)-ATPase in the regulation of pHi homoeostasis in T. cruzi.

Phosphatidylethanol is formed by "transphosphatidylation' of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca(2+)-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca(2+)-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidyl-alcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca(2+)-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca(2+) concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca(2+)-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

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] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma-membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps.

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.

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.