Molecular features of copper binding proteins involved in copper homeostasis.

Copper has a wide and important role in biological systems, determining conformation and activity of many metalloproteins and enzymes, such as cytochrome oxidase and superoxide dismutase . Furthermore, due to its possible reactivity with nonspecific proteins and toxic effects, elaborate systems of absorption, concentration buffering, delivery to specific protein sites and elimination, require a complex system including small carriers, chaperones and active transporters. The P-type copper ATPases ATP7A and ATP7B provide an important system for acquisition, active transport, distribution and elimination of copper. Relevance of copper metabolism to human diseases and therapy is already known. It is quite certain that further studies will reveal detailed and useful information on biochemical mechanisms and relevance to diseases. © 2016 IUBMB Life, 69(4):211-217, 2017.

Biochemical characterization of P-type copper ATPases.

Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and transmembrane helices containing cation-binding sites [TMBS (transmembrane metal-binding sites)] for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H+ exchange. Although PII-type ATPases sustain active transport of alkali/alkali-earth ions (i.e. Na+, Ca2+) against electrochemical gradients across defined membranes, PIB-type ATPases transfer transition metal ions (i.e. Cu+) from delivery to acceptor proteins and, prominently in mammalian cells, undergo trafficking from/to various membrane compartments. A specific component of copper ATPases is the NMBD (N-terminal metal-binding domain), containing up to six copper-binding sites in mammalian (ATP7A and ATP7B) enzymes. Copper occupancy of NMBD sites and interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper.

Translocation of platinum anticancer drugs by human copper ATPases ATP7A and ATP7B.

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance. Down-regulation of CTR1 and up-regulation of the Cu-ATPases, ATP7A and ATP7B, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu-ATPases, we performed electrical measurements on the COS-1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, and adsorbed on a solid supported membrane. The experimental results indicate that Pt drugs activate Cu-ATPases and undergo ATP-dependent translocation in a fashion similar to that of Cu. We then used NMR spectroscopy and ESI-MS to determine the binding mode of these drugs to the first N-terminal metal-binding domain of ATP7A (Mnk1).

Ca(2+)/H (+) exchange, lumenal Ca(2+) release and Ca (2+)/ATP coupling ratios in the sarcoplasmic reticulum ATPase.

The Ca(2+) transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca(2+) in intracellular membrane bound compartments, thereby lowering cytosolic Ca(2+) to induce relaxation. The stored Ca(2+) is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca(2+), whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca(2+). We review here biochemical and biophysical evidence demonstrating that release of bound Ca(2+) into the lumen of SR requires Ca(2+)/H(+) exchange at the low affinity Ca(2+) sites. Rise of lumenal Ca(2+) above its dissociation constant from low affinity sites, or reduction of the H(+) concentration by high pH, prevent Ca(2+)/H(+) exchange. Under these conditions Ca(2+) release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca(2+)pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.

Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state.

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca(2+)-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca(2+) pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates. An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca(2+)-binding sites ready to accept new cytosolic Ca(2+). In the absence of Ca(2+) and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca(2+)), and if millimolar Mg(2+) is present, one Mg(2+) is expected to occupy one of the Ca(2+)-binding sites with a millimolar dissociation constant. This Mg(2+) accelerates the reaction cycle, not permitting phosphorylation without Ca(2+) binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg(2+) state at 3.0 Å resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin, a small regulatory membrane protein of Ca(2+)-ATPase, is bound, stabilizing the E1·Mg(2+) state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of ß-adrenergic signal in Ca(2+) regulation in heart (for reviews, see, for example, refs 8-10), and seems to play an important role in muscle-based thermogenesis. We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump.

Distinctive features of catalytic and transport mechanisms in mammalian sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases.

Ca(2+) (sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA)) and Cu(+) (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca(2+), demonstrated by the addition of ATP and Ca(2+) to SERCA deprived of Ca(2+) (E2) as compared with ATP to Ca(2+)-activated enzyme (E1·2Ca(2+)). Activation by Ca(2+) is slower at low pH (2H(+)·E2 to E1·2Ca(2+)) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca(2+) translocation. A "H(+)-gated pathway," demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca(2+) release by H(+)/Ca(2+) exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu(+)/H(+) exchange. As opposed to SERCA after Ca(2+) chelation, ATP7A/B does not undergo reverse phosphorylation with P(i) after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.

Analysis of calcium transients in cardiac myocytes and assessment of the sarcoplasmic reticulum Ca2+-ATPase contribution.

Ca(2+) signaling plays an essential role in several functions of cardiac myocytes. Transient rises and reductions of cytosolic Ca(2+), permitted by the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) and other proteins, control each cycle of contraction and relaxation. Here we provide a practical method for isolation of neonatal rat cardiac myocytes and measurement of Ca(2+) transients in cultured cardiac myocytes, yielding information on kinetic resolution of the transients, variations of cytosolic Ca(2+) concentrations, and adequacy of intracellular Ca(2+) stores. We also provide examples of experimental perturbations that can be used to assess the contribution of SERCA2 to Ca(2+) signaling.

Regulation and rate limiting mechanisms of Ca2+ ATPase (SERCA2) expression in cardiac myocytes.

Involvement of the calcineurin/NFAT pathway in transcription of cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) was demonstrated (Prasad and Inesi, Am J Physiol Heart Circ Physiol 300(1):H173-H180, 2011) by upregulation of SERCA2 following calcineurin (CN) activation by cytosolic Ca(2+), and downregulation of SERCA2 following CN inhibition with cyclosporine (CsA) or CN subunits gene silencing. We show here that in cultured cardiac myocytes, competitive engagement of the CN/NFAT pathway is accompanied by downregulation of SERCA2 and Ca(2+) signaling alterations. In fact, SERCA2 downregulation occurs following infection of myocytes with adenovirus vectors carrying luciferase or SERCA1 cDNA under control of NFAT-dependent promoters, but not under control of CMV promoters that do not depend on NFAT. SERCA2 downregulation is demonstrated by comparison with endogenous transcription and protein expression standards such as GAPDH and actin, indicating prominent SERCA2 involvement by the CN/NFAT pathway. Transcription of genes involved in hypertrophy, triggered by adrenergic agonist or by direct protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), is also prominently dependent on CN/NFAT. This is demonstrated by CN inhibition with CsA, CN subunits gene silencing with siRNA, displacement of NFAT from CN with 9,10-Dihydro-9,10[1',2']-benzenoanthracene-1,4-dione (INCA-6), and myocyte infection with vectors carrying luciferase cDNA under control of NFAT-dependent promoter. We show here that competitive engagement of the CN/NFAT pathway by endogenous genes involved in hypertrophy produces downregulation of SERCA2, reduction of Ca(2+) transport and inadequate Ca(2+) signaling. It is most interesting that, in the presence of adrenergic agonist, specific protein kinase C (PKC) inhibition with 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (Gö 6983) prevents development of hypertrophy and maintains adequate SERCA2 levels and Ca(2+) signaling.

The Ca2+-ATPase (SERCA1) is inhibited by 4-aminoquinoline derivatives through interference with catalytic activation by Ca2+, whereas the ATPase E2 state remains functional.

Several clotrimazole (CLT) and 4-aminoquinoline derivatives were synthesized and found to exhibit in vitro antiplasmodial activity with IC(50) ranging from nm to µm values. We report here that some of these compounds produce inhibition of rabbit sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) with IC(50) values in the µm range. The highest affinity for the Ca(2+)-ATPase was observed with NF1442 (N-((3-chlorophenyl)(4-((4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)phenyl)methyl)-7-chloro-4-aminoquinoline) and NF1058 (N-((3-chlorophenyl)(4-(pyrrolidin-1-ylmethyl)phenyl)methyl)-7-chloro-4-aminoquinoline),yielding IC(50) values of 1.3 and 8.0 µm as demonstrated by measurements of steady state ATPase activity as well as single cycle charge transfer. Characterization of sequential reactions comprising the ATPase catalytic and transport cycle then demonstrated that NF1058, and similarly CLT, interferes with the mechanism of Ca(2+) binding and Ca(2+)-dependent enzyme activation (E(2) to E(1)·Ca(2) transition) required for formation of phosphorylated intermediate by ATP utilization. On the other hand, Ca(2+) independent phosphoenzyme formation by utilization of P(i) (i.e. reverse of the hydrolytic reaction in the absence of Ca(2+)) was not inhibited by NF1058 or CLT. Comparative experiments showed that the high affinity inhibitor thapsigargin interferes not only with Ca(2+) binding and phosphoenzyme formation with ATP but also with phosphoenzyme formation by utilization of P(i) even though this reaction does not require Ca(2+). It is concluded that NF1058 and CLT inhibit SERCA by stabilization of an E(2) state that, as opposed to that obtained with thapsigargin, retains the functional ability to form E(2)-P by reacting with P(i).

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms.

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca(2+) with high affinity on one side of the membrane, and releases the bound Ca(2+) on the other side of the membrane to yield a high free Ca(2+) gradient. These features are a basic requirement for cellular Ca(2+) signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.

Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase).

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.

Silencing calcineurin A subunit reduces SERCA2 expression in cardiac myocytes.

Resting intracellular Ca(2+) can be raised, in neonatal rat cardiac myocytes, by exposure to very low concentration of thapsigargin (TG). Such a Ca(2+) rise yields calcineurin (CN) activation demonstrated by increased expression of transfected luciferase cDNA under control of nuclear factor of activated T-cells (NFAT) promoter and increased translocation of NFAT to nuclei. We found that exposure of cardiac myocytes to TG is followed by increase of sarcroplasmic reticulum Ca(2+) transport ATPase (SERCA2) expression, which is further increased when CN inactivation by CAMKII (calmodulin-dependent kinase) is prevented with KN93 (CAMKII inhibitor). On the other hand, SERCA2 expression is reduced by CN inhibition with cyclosporine. We have now induced calcineurin A (CNA) α- or ß-subunit gene silencing with small interfering RNA (siRNA) and observed strong interference with expression of SERCA2, both in control myocytes and following exposure to TG. Such interference is also obtained following NFAT displacement from CN with 9,10-dihydro-9,10[1',2']-benzenoanthracene-1,4-dione (INCA-6). We have also observed analogous effects on expression of phospholamban (PLB) and Na(+)/Ca(2+) exchanger (NCX). Pertinent to these findings, we have identified, by in-silico analysis, NFAT binding sites in SERCA2, PLB, and NCX1 promoters. Our experiments indicate that activation of the calcineurin-NFAT pathway by rise of resting cytosolic Ca(2+) elevates transcription/expression of SERCA2, PLB, and NCX1, providing a homeostatic mechanism for long-term control of cytosolic Ca(2+).

ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements.

ATP7B is a copper dependent P-type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate. We attribute this charge displacement to movement of bound copper within ATP7B. Based on specific mutations, we demonstrate that enzyme activation by copper requires occupancy of a site in the N-terminus extension which is not present in other transport ATPases, as well as of a transmembrane site corresponding to the cation binding site of other ATPases.

Comparative features of copper ATPases ATP7A and ATP7B heterologously expressed in COS-1 cells.

ATP7A and ATP7B are P-type ATPases required for copper homeostasis and involved in the etiology of Menkes and Wilson diseases. We used heterologous expression of ATP7A or ATP7B in COS-1 cells infected with adenovirus vectors to characterize differential features pertinent to each protein expressed in the same mammalian cell type, rather than to extrinsic factors related to different cells sustaining expression. Electrophoretic analysis of the expressed protein, before and after purification, prior or subsequent to treatment with endoglycosidase, and evidenced by protein or glycoprotein staining as well as Western blotting, indicates that the ATP7A protein is glycosylated while ATP7B is not. This is consistent with the prevalence of glycosylation motifs in the ATP7A sequence, and not in ATP7B. ATP7A and ATP7B undergo copper-dependent phosphorylation by utilization of ATP, forming equal levels of an "alkali labile" phosphoenzyme intermediate that undergoes similar catalytic (P-type ATPase) turnover in both enzymes. In addition, incubation with ATP yields an "alkali stable" phosphoprotein fraction, attributed to phosphorylation of serines. Alkali stable phosphorylation occurs at lower levels in ATP7A, consistent with a different distribution of serines in the amino acid sequence. Immunostaining of COS-1 cells sustaining heterologous expression shows initial association of both ATP7A and ATP7B with Golgi and the trans-Golgi network. However, in the presence of added copper, ATP7A undergoes prevalent association with the plasma membrane while ATP7B exhibits intense trafficking with cytosolic vesicles. Glycosylation of ATP7A and phosphorylation of ATP7B apparently contribute to their different trafficking and membrane association when expressed in the same cell type.

High yield heterologous expression of wild-type and mutant Cu+-ATPase (ATP7B, Wilson disease protein) for functional characterization of catalytic activity and serine residues undergoing copper-dependent phosphorylation.

ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7B in COS1 cells infected with adenovirus vector. ATP7B, isolated with the microsomal fraction of cell homogenates, accounts for 10-20% of the total protein. Copper-dependent, steady-state ATPase yields 30 nmol of P(i)/mg of protein/min at 37 degrees C, pH 6.0. ATP7B phosphorylation with ATP occurs with diphasic kinetics and is totally copper-dependent. Alkali labile phosphoenzyme (catalytic intermediate of P-ATPases) accounts for a small fraction of the total phosphoprotein and is prevented by D1027N (P domain) or C983A/C985A (CXC copper binding motif in TM6) mutations. Decay of [(32)P]phosphoenzyme following chase with non-radioactive ATP occurs with an initial burst involving alkali labile phosphoenzyme (absent in D1027N and C983A/C985A mutants) and continues at a slow rate involving alkali-resistant phosphoenzyme. If a copper chelator is added with the ATP chase, the initial burst is smaller, and further cleavage is totally inhibited. Analysis by proteolysis and mass spectrometry demonstrates that the alkali stable phosphoenzyme involves Ser(478) and Ser(481) (NMBD), Ser(1121) ("N" domain) and Ser(1453) (C terminus), and occurs with the same pattern ex vivo (COS-1) and in vitro (microsomes). The overall copper dependence of phosphorylation and hydrolytic cleavage suggests long range conformational effects, including interactions of NMBD and headpiece domains, with strong influence on catalytic turnover.

High-yield heterologous expression of wild type and mutant Ca(2+) ATPase: Characterization of Ca(2+) binding sites by charge transfer.

High-yield heterologous SERCA1 (Ca(2+) ATPase) expression was obtained in COS-1 cells infected with recombinant adenovirus vector (rAdSERCA). Higher transcription and expression were obtained in the presence of a His(6) tag at the amino terminus, as compared with a His(6) tag at the carboxyl SERCA terminus, or no tag. The expressed protein was targeted extensively to intracellular membranes. Optimal yield of functional Ca(2+) ATPase corresponded to 10% of total protein, with phosphoenzyme levels, catalytic turnover and Ca(2+) transport identical with those of native SERCA1. This recombinant membrane-bound (detergent-free) enzyme was used for characterization of Ca(2+) binding at the two specific transmembrane sites (ATP-free) by measurements of net charge transfer upon Ca(2+) binding to the protein, yielding cooperative isotherms (K(1)=5.9+/-0.5x10(5) M(-1) and K(2)=5.7+/-0.3x10(6) M(-1)). Non-cooperative binding of only one Ca(2+), and loss of ATPase activation, were observed following E309 mutation at site II. On the other hand, as a consequence of the site II mutation, the affinity of site I for Ca(2+) was increased (K=4.4+/-0.2x10(6) M(-1)). This change was due to a pK(a) shift of site I acidic residues, and to contributions of oxygen functions from empty site II to Ca(2+) binding at site I. No charge movement was observed following E771Q mutation at site I, indicating no Ca(2+) binding to either site. Therefore, calcium occupancy of site I is required to trigger cooperative binding to site II and catalytic activation. In the presence of millimolar Mg(2+), the charge movement upon addition of Ca(2+) to WT ATPase was reduced by 50%, while it was reduced by 90% when Ca(2+) was added to the E309Q/A mutants, demonstrating that competitive Mg(2+) binding can occur at site I but not at site II.

Reaction cycle of Thermotoga maritima copper ATPase and conformational characterization of catalytically deficient mutants.

Copper transport ATPases sustain important roles in homeostasis of heavy metals and delivery of copper to metalloenzymes. The copper transport ATPase from Thermotoga maritima (CopA) provides a useful system for mechanistic studies, due to its heterologous expression and stability. Its sequence comprises 726 amino acids, including the N-terminal metal binding domain (NMBD), three catalytic domains (A, N, and P), and a copper transport domain formed by eight helices, including the transmembrane metal binding site (TMBS). We performed functional characterization and conformational analysis by proteolytic digestion of WT and mutated (NMBD deletion or mutation) T. maritima CopA, comparing it with Archaeoglobus fulgidus CopA and Ca(2+) ATPase. A specific feature of T. maritima CopA is ATP utilization in the absence of copper, to form a low-turnover phosphoenzyme intermediate, with a conformation similar to that obtained by phosphorylation with P(i) or phosphate analogues. On the other hand, formation of an activated state requires copper binding to both NMBD and TMBS, with consequent conformational changes involving the NMBD and A domain. Proteolytic digestion analysis demonstrates A domain movements similar to those of other P-type ATPases to place the conserved TGES motif in the optimal position for catalytic assistance. We also studied an H479Q mutation (analogous to one of human copper ATPase ATP7B in Wilson disease) that inhibits ATPase activity. We found that, in spite of the H479Q mutation within the nucleotide binding domain, the mutant still binds ATP, yielding a phosphorylation transition state conformation. However, covalent phosphoryl transfer is not completed, and no catalytic turnover is observed.

Effects of thapsigargin and phenylephrine on calcineurin and protein kinase C signaling functions in cardiac myocytes.

Neonatal rat cardiac myocytes were exposed to 10 nM thapsigargin (TG) or 20 muM phenylephrine (PE) to compare resulting alterations of Ca(2+) homeostasis. Either treatment results in resting cytosolic [Ca(2+)] rise and reduction of Ca(2+) signals in myocytes following electrical stimuli. In fact, ATP-dependent Ca(2+) transport is reduced due to catalytic inhibition of sarcoplasmic reticulum ATPase (SERCA2) by TG or reduction of SERCA2 protein expression by PE. A marked rise of nuclear factor of activated T cells (NFAT)-dependent expression of transfected luciferase cDNA is produced by TG or PE, which is dependent on increased NFAT dephosphorylation by activated calcineurin and reduced phosphorylation by inactivated glycogen synthase kinase 3beta. Expression of SERCA2 (inactivated) protein is increased following exposure to TG, whereas no hypertrophy is produced. On the contrary, SERCA2 expression is reduced, despite high CN activity, following protein kinase C (PKC) activation by PE (or phorbol 12-myristate 13-acetate) under conditions producing myocyte hypertrophy. Both effects of TG and PE are dependent on NFAT dephosphorylation by CN, as demonstrated by CN inhibition with cyclosporine (CsA). However, the hypertrophy program triggered by PKC activation bypasses SERCA2 transcription and expression due to competitive recruitment of NFAT and/or other transcriptional factors. A similar dependence on CN activation, but relative reduction under conditions of PKC activation, involves transcription and expression of the Na(+)/Ca(2+) exchanger-1. On the other hand, significant upregulation of transient receptor potential channel proteins is noted following PKC activation. The observed alterations of Ca(2+) homeostasis may contribute to development of contractile failure.

Intermediate phosphorylation reactions in the mechanism of ATP utilization by the copper ATPase (CopA) of Thermotoga maritima.

Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78-2.73 micromol/mg/min in the presence of Cu+ (pH 6, 60 degrees C) and 0.03-0.08 micromol/mg/min in the absence of Cu+. High levels of enzyme phosphorylation are obtained by utilization of [gamma-32P]ATP in the absence of Cu+. This phosphoenzyme decays at a much slower rate than observed with Cu.E1 approximately P. In fact, the phosphoenzyme is reduced to much lower steady state levels upon addition of Cu+, due to rapid hydrolytic cleavage. Negligible ATPase turnover is sustained by CopA following deletion of its N-metal binding domain (DeltaNMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [gamma-3)P]ATP by the DeltaNMBD and CXXC mutants, with no effect of Cu+ either on its formation or hydrolytic cleavage. Phosphoenzyme formation (E2P) can also be obtained by utilization of Pi, and this reaction is inhibited by Cu+ (E2 to E1 transition) even in the DeltaNMBD mutant, evidently due to Cu+ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in DeltaNMBD and slower in CXXC mutant. We propose that Cu+ binding to the NMBD is required to produce an "active" conformation of CopA, whereby additional Cu+ bound to an alternate (transmembrane transport) site initiates faster cycles including formation of Cu.E1 approximately P, followed by the E1 approximately P to E2-P conformational transition and hydrolytic cleavage of phosphate. An H479Q mutation (analogous to one found in Wilson disease) renders CopA unable to utilize ATP, whereas phosphorylation by Pi is retained.