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Stimulatory and inhibitory effects of inorganic lead on calcineurin
Toxicology 150 (2000) 171 – 178 www.elsevier.com/locate/toxicol
Stimulatory and inhibitory effects of inorganic lead on calcineurin Marcey Kern, Gerald Audesirk * Biology Department, Uni6ersity of Colorado at Den6er, PO Box 173364, Den6er, CO 80217 -3364, USA Received 10 February 2000; accepted 14 June 2000
Abstract Calcineurin is a phosphatase with activity dependent on both Ca2 + /calmodulin binding to the catalytic A subunit and Ca2 + binding to the regulatory B subunit. We have previously shown that Pb2 + activates calmodulin with a threshold of about 100 pM free Pb2 + , and that Pb2 + and Ca2 + are roughly additive in calmodulin activation (Kern et al., NeuroToxicology 21, 353–364 (2000)). In the present study, we evaluated the effects of Pb2 + , with and without Ca2 + and calmodulin, on calcineurin activity. In calmodulin-containing, Ca2 + -free solutions, Pb2 + activated calcineurin with a threshold of about 100 pM free Pb2 + . Maximum calcineurin activity (comparable to that induced by 10 mM Ca2 + ) was reached at about 200 pM free Pb2 + . Higher Pb2 + concentrations reduced activity, although some activity remained even at 2000 pM free Pb2 + . Combined with subsaturating Ca2 + concentrations, as little as 20 pM free Pb2 + enhanced calcineurin activity, but free Pb2 + concentrations greater than 200 pM still reduced activity below maximum. Extremely high Ca2 + concentrations (10 mM) completely reversed the inhibition of activity by 2000 pM free Pb2 + . In the absence of calmodulin, Ca2 + slightly stimulated calcineurin activity. Pb2 + did not substitute for Ca2 + in calmodulin-free activation; in fact, high concentrations of Pb2 + inhibited Ca2 + -mediated activation. We tentatively conclude that low concentrations of free Pb2 + activate calcineurin by activating calmodulin. Higher concentrations reduce calcineurin activity, perhaps by binding to the B subunit. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calcineurin; Protein phosphatase 2B; Lead; Pb2 + , calcium; Ca2 +
1. Introduction Calcineurin (protein phosphatase 2B) is a serine/threonine protein phosphatase with wide* Corresponding author. Tel.: +1-303-5562593; fax: +1303-5564352. E-mail address: [email protected] (G. Audesirk).
spread distribution in the brain, immune system, and other tissues. The phosphatase activity of calcineurin is dependent on both Ca2 + /calmodulin binding to the catalytic A subunit and Ca2 + binding to the regulatory B subunit (Stewart et al., 1982; Klee et al., 1988; Stemmer and Klee, 1994; Perrino et al., 1995; Perrino and Soderling, 1998). Inorganic lead (Pb2 + ) binds to many Ca2 + -binding proteins, where it may act as an agonist,
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 2 5 8 - 4
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antagonist, or partial agonist. For example, Pb2 + acts as an agonist in activating calmodulin (Goldstein and Ar, 1983; Habermann et al., 1983; Kern et al., 2000), as an antagonist in inhibiting Ca2 + -dependent inactivation of voltage-sensitive calcium channels (Sun and Suszkiw, 1995), and as a partial agonist in activating Ca2 + -dependent isoforms of protein kinase C (Long et al., 1994; Tomsig and Suszkiw, 1995; but see also Saijoh et al., 1988; Murakami et al., 1993). We have previously shown that Pb2 + is a full agonist in activation of calmodulin, as assayed by calmodulin-dependent stimulation of cyclic nucleotide phosphodiesterase (PDE) and stimulation of the fluorescence of hydrophobic probes (Kern et al., 2000). The threshold for calmodulin activation in both assays is about 100 pM free Pb2 + . Probably of more physiological importance, Pb2 + and Ca2 + are roughly additive in activating calmodulin-dependent stimulation of PDE. Therefore, we hypothesized that Pb2 + would act as an agonist for calmodulin-dependent stimulation of calcineurin activity, through Pb2 + / calmodulin binding to the catalytic A subunit. Further, we predicted that Pb2 + and Ca2 + would be roughly additive in calmodulin-dependent stimulation of calcineurin activity. The possible effects of Pb2 + binding to the EF hands of the B subunit (Kakalis et al., 1995) could not be predicted. Further, calcineurin apparently has at least one cysteine in the A subunit that affects enzymatic activity (King, 1986). Because Pb2 + has a high affinity for cysteine residues (Al-Modhefer et al., 1991), Pb2 + might also alter calcineurin activity by binding to this cysteine residue. Overall, then, the effects of Pb2 + on calcineurin activity could range from simple activation to complex, possibly concentration-dependent stimulatory and inhibitory actions. We evaluated the effects of Pb2 + , with and without Ca2 + and calmodulin, on calcineurin activity. In the presence of calmodulin, low concentrations of free Pb2 + ( 5200 pM) stimulated calcineurin activity, but higher Pb2 + concentrations reduced activity. Stimulation of calcineurin activity by combinations of Ca2 + and low Pb2 + concentrations was roughly additive. High Ca2 + concentrations reversed the reduction in activity
caused by high Pb2 + concentrations. Ca2 + , but not Pb2 + , supported a low level of calcineurin activity in the absence of calmodulin.
2. Methods
2.1. Materials Calcineurin was purchased from Boehringer Mannheim, calmodulin from Calbiochem, EGTA from J.T. Baker, and a phosphorylated peptide corresponding to a portion of the RII subunit of cyclic AMP-dependent protein kinase (DonellaDeana et al., 1994) from Biomol. Calcineurin phosphatase activity was measured as phosphate released from the RII subunit. Phosphate was measured by its reaction with molybdate/malachite green (Lanzetta et al., 1979), using the reagents of the serine/threonine phosphatase assay kit from Promega. All other chemicals were purchased from Sigma.
2.2. Assay methods The reaction mixture (100 ml final volume) consisted of 100 nM calcineurin, 750 nM calmodulin, 25 mM phosphorylated RII peptide, 2% trehalose, 1 mM EGTA, 10 mM MgCl2, and 25 mM Tris (pH 7.3), with CaCl2 or Pb(NO3)2 added to produce solutions with variable concentrations of free Ca2 + and/or free Pb2 + ions. For experiments in which high concentrations of Ca2 + and Pb2 + were needed (see Fig. 3), the buffer also contained 1 mM nitrilotriacetic acid (NTA). All solutions were checked daily for phosphate contamination. Free metal concentrations were estimated using the Chelator program (Schoenmakers et al., 1992) and the stability constants for ligand/metal complexes given in Martell and Smith (1974). The stability constants used for EGTA/metal complexes (all given for 20°C and 0.1 M ionic strength) were (1) Pb2 + /EGTA: 14.71; (2) Ca2 + / EGTA: 10.97; and (3) Mg2 + /EGTA: 5.21. The stability constants for NTA/metal complexes were (1) Pb2 + /NTA: 11.39; (2) Ca2 + /NTA: 6.41; and (3) Mg2 + /NTA: 5.41. The Chelator program corrects the stability constants for other temperatures
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and ionic strengths. Other stability constants (e.g. for Pb2 + /EGTA: 13.48 (Simons 1995)) give slightly higher free metal ion concentrations than those given in the Results, but do not affect the interpretation of the experiments. To measure the phosphatase activity of calcineurin, all of the ingredients except the RII peptide were mixed together and the reaction was started by rapid addition of the RII peptide. The
Fig. 1. Calcineurin activity as a function of free Ca2 + and/or Pb2 + ion concentrations. All points in all figures represent mean9 s.e.m. of at least six replicates in at least three different experiments. (a) Calcineurin activity in the presence of Ca2 + alone (filled circles) or variable Ca2 + plus 100 pM free Pb2 + (open circles). The curves are four-parameter Hill equations fit to the data points. The EC50 for activation by Ca2 + alone was approximately 740 950 nM; in the presence of 100 pM free Pb2 + , the EC50 for activation by Ca2 + was reduced to approximately 340 930 nM. (b) Calcineurin activity in the presence of Pb2 + alone (filled circles) or variable Pb2 + plus 500 nM free Ca2 + (open circles). Between 20 and 100 pM free Pb2 + , the combination of Pb2 + and Ca2 + stimulated significantly greater calcineurin activity than was stimulated by either cation alone. Between 200 and 2000 pM free Pb2 + , calcineurin activity was not affected by 500 nM Ca2 + .
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reaction mixture was incubated at 30°C for 30 min, then the reaction was stopped by adding 100 ml of the Promega molybdate/malachite green dye mixture. The resulting solution was incubated at room temperature for 20 min, after which the absorbance was measured at 600 nm. Preliminary experiments showed that this incubation period was within the linear range of both calcineurin activity (at this substrate concentration) and the molybdate/malachite green phosphate measurement. Calcineurin activity in the absence of calmodulin was measured with the same protocol but with calmodulin omitted and the incubation period increased to 2 h. All data points shown in the figures represent means 9s.e.m. of at least six replicates in at least three separate experiments. All calcineurin activity measurements given in the Results were corrected by subtracting the absorbance of reaction mixtures containing RII peptide but with calcineurin omitted. Ca2 + - and Pb2 + -independent activity (in the presence of calmodulin) varied in different experiments from about 3 to 15% of the activity in the presence of saturating calmodulin and Ca2 + . Phosphate release was estimated by comparison with standards of known phosphate concentration. Maximum calcineurin activity in the presence of excess calmodulin and saturating free Ca2 + was : 2.59 0.3 (mean 9 s.e.m.) picomoles of phosphate released per minute per picomole calcineurin. This is equivalent to about 31 pm/ min/mg, which is reasonably consistent with the 57 pm/min/mg determined by Donella-Deana et al. (1994) using the same substrate but a different buffer and pH 8. For ease of comparison, calcineurin activity in the Results is presented as a fraction of the activity in solutions containing saturating calmodulin and free Ca2 + (10 mM or 1 mM; see Fig. 1).
2.3. Statistical analysis Activation curves of calcineurin activity as a function of free cation concentration were generated by fitting the data to a four-parameter Hill plot (Sigma Plot, SPSS). The curve fits were then used to estimate thresholds and EC50 values. Where appropriate, data were also analyzed by
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ANOVA, followed by Dunnett’s test for multiple comparisons between a single control and several experimental conditions, or by the Student – Neumann–Keuls test for multiple comparisons among treatments. Effects were considered significant at the 0.05 level.
3. Results
3.1. Acti6ation of calcineurin by Ca 2 + and Pb 2 + Calcineurin activity was measured in nominally Ca2 + -free conditions (1 mM EGTA, no added Ca2 + ) and in solutions containing approximate free Ca2 + concentrations of 50 nM to 1 mM (filled circles in Fig. 1a). Ca2 + -dependent activity was statistically significant at 500 nM free Ca2 + and saturated at 2– 10 mM free Ca2 + . The EC50 for calcineurin activation was :740 9 50 nM free Ca2 + (mean9s.e.m.). This EC50 concentration is similar to that reported in the literature (Stemmer and Klee, 1994). Calcineurin activity was similarly measured in nominally Pb2 + -free conditions (1 mM EGTA, no added Pb2 + ) and in solutions containing approximate free Pb2 + concentrations of 10 – 2000 pM (filled circles in Fig. 1b). Pb2 + activated calcineurin activity at concentrations of 100 pM and above. Calcineurin activity peaked at about 200 pM free Pb2 + at a level that was not significantly different from the activity in the presence of saturating free Ca2 + (P \ 0.5). Free Pb2 + concentrations above 200 pM reduced calcineurin activity, but some phosphatase activity remained even at 2000 pM free Pb2 + .
3.2. Interactions between Ca 2 + and Pb 2 + in calcineurin acti6ation Pb2 + and Ca2 + are roughly additive in activation of calmodulin (Kern et al., 2000). Therefore, we predicted that Pb2 + and Ca2 + should act additively in activation of calcineurin. We tested the effects of suboptimal concentrations of free Pb2 + in the presence of variable free Ca2 + , and vice versa.
First, calcineurin activity was measured in solutions containing a constant 100 pM free Pb2 + and concentrations of free Ca2 + varying between 100 nM and 10 mM. This concentration of free Pb2 + was chosen because it supports low calcineurin activity (Fig. 1b), and therefore would enable us to detect positive or negative interactions between Ca2 + and Pb2 + . One hundred pM free Pb2 + augmented calcineurin activation by Ca2 + , lowering the free Ca2 + threshold and shifting Ca2 + activation curve to the left; i.e. lower concentrations of free Ca2 + were needed to activate calcineurin in the presence of Pb2 + (open circles in Fig. 1a). The EC50 for activation by Ca2 + in the presence of 100 pM free Pb2 + was : 340+ 30 nM free Ca2 + , compared to 740 nM free Ca2 + in the absence of Pb2 + . The maximum calcineurin activity remained the same at high free Ca2 + concentrations with or without 100 pM free Pb2+. Second, we tested the effects of variable free Pb2 + concentrations on the activation of calcineurin by 500 nM free Ca2 + , a Ca2 + concentration that supports modest calcineurin activity (see Fig. 1a). Low concentrations of free Pb2 + (i.e. between 20 and 100 pM) increased calcineurin activity compared to that caused by 500 nM free Ca2 + alone (open circles in Fig. 1b). Maximum calcineurin activity still occurred with about 200 pM free Pb2 + , and this activity was not significantly different from that caused by saturating free Ca2 + (P\0.5). The reduction of calcineurin activity caused by high concentrations of free Pb2 + (over 200 pM) was not affected by 500 nM free Ca2 + (Fig. 1b). This might indicate that the reduction is caused by binding of Pb2 + to a site on calcineurin that does not bind Ca2 + . However, Pb2 + has much higher affinity than Ca2 + for many Ca2 + -binding proteins (e.g. Markovac and Goldstein, 1988; Long et al., 1994; Tomsig and Suszkiw, 1995; Kern et al., 2000). Therefore, it is also possible that Pb2 + at concentrations above 200 pM may simply outcompete 500 nM free Ca2 + for a site on calcineurin for which Ca2 + is either permissive or stimulatory, and inhibit enzyme activity. If so, then very high free Ca2 + concentrations should be able to reverse the reduction of activity caused
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not support calmodulin-independent calcineurin activity at any concentration from 100 pM to 50 nM (data not shown). Further, Pb2 + reduced (2 nM) or eliminated (50 nM) the stimulation of calmodulin-independent activity by 10 mM Ca2 + (Fig. 3). Finally, 10 mM Ca2 + supported full calmodulin-dependent activity in the presence of 2 nM Pb2 + (Fig. 2) but did not support full calmodulinindependent activity in the presence of 2 nM Pb2 + (Fig. 3). This apparent discrepancy probably results from the fact that calmodulin-dependent calcineurin activity was over 30-fold greater than calmodulin-independent activity. Therefore, in the presence of calmodulin (Fig. 2), a small inhibition of activity caused by calmodulin-independent effects of Pb2 + could not be detected against the high background of calmodulin-dependent activity stimulated by Ca2 + and Pb2 + . Fig. 2. The reduced calcineurin activity caused by 2 nM free Pb2 + was completely reversed by 10 mM free Ca2 + . All data points, including 10 mM free Ca2 + and 2 nM free Pb2 + , represent new experiments (i.e. the data points are not repeated from earlier figures). Asterisk: significantly different from activity with 10 mM Ca2 + .
by Pb2 + . To test this hypothesis, we compared calcineurin activity in the presence of 10 mM free Ca2 + alone (a saturating concentration; see Fig. 1a), 2 nM free Pb2 + alone, or both in combination (Fig. 2). We found that 10 mM free Ca2 + reversed the activity reduction caused by 2 nM free Pb2 + .
3.3. Effects of Ca 2 + and Pb 2 + on calmodulin-independent calcineurin acti6ity Calcineurin has limited activity in the absence of calmodulin (Haddy et al., 1992; Perrino et al., 1995). We examined the effects of Ca2 + and Pb2 + , alone and in combination, on this calmodulin-independent activity. In agreement with many reports in the literature, we found that significant calmodulin-free calcineurin activity required Ca2 + (Fig. 3). Calmodulin-independent activity in the presence of 10 mM free Ca2 + was about 3% of the activity that occurred with supersaturating calmodulin concentrations. Pb2 + did
Fig. 3. The effects of Ca2 + and Pb2 + on calmodulin-independent calcineurin activity. Phosphatase activity was not significantly different from zero in the absence of either cation (black bar). Saturating free Ca2 + (10mM) in calmodulin-free solutions (open bar) stimulated about 3% of the maximum activity seen in solutions containing both saturating calmodulin and Ca2 + . Pb2 + reduced the Ca2 + -dependent, calmodulin-independent activity (striped bars). Asterisks: significantly different from activity with 10 mM Ca2 + .
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4. Discussion
4.1. Stimulatory and inhibitory effects of Pb 2 + on calcineurin acti6ity The activity of calcineurin is controlled by a complex interaction among several factors, including calmodulin binding to the A (catalytic) subunit, the binding of Ca2 + to calmodulin and independently to the B subunit (Klee et al., 1988; Stemmer and Klee, 1994; Perrino et al., 1995; Perrino and Soderling, 1998), the presence and oxidation state of iron and zinc in the active site (King and Huang, 1986; Kissinger et al., 1995; Wang et al., 1996), and the oxidation state of a critical cysteine in the A subunit (King, 1986). We found that Pb2 + had both stimulatory and inhibitory effects on calcineurin activity (Fig. 1b). In principle, these effects might be caused by interactions of Pb2 + with calmodulin or with any of several sites on calcineurin itself. We had previously shown that Pb2 + activated calmodulin, with a threshold of about 100 – 300 pM free Pb2 + (Kern et al., 2000). High free Pb2 + concentrations (from about 1000 pM up to 5000 pM, the highest concentration tested) activated calmodulin to the same extent as saturating Ca2 + . Further, Pb2 + and Ca2 + were additive in calmodulin activation. Finally, there were no inhibitory effects of any concentration of Pb2 + , with or without Ca2 + . Therefore, Pb2 + appears to be a full agonist for calmodulin activation. In the present study, low concentrations of Pb2 + stimulated calcineurin activity (Fig. 1b), the maximum activity obtained with Pb2 + alone (at 200 pM) did not differ from the maximum activity obtained with saturating concentrations of Ca2 + , and low concentrations of Pb2 + were roughly additive with Ca2 + in activating calcineurin, all of which would be expected of Pb2 + activation of calmodulin. No concentration of Pb2 + from 100 pM through 50 nM was able to support significant calmodulin-independent activity, which would be expected if Pb2 + stimulated calcineurin activity by a direct interaction between Pb2 + and calcineurin itself. Therefore, we conclude that Pb2 + stimulates calcineurin by activation of calmodulin.
There was progressively less calcineurin activation with free Pb2 + concentrations above 200 pM, although there was still some stimulation of calcineurin activity even at 2000 pM free Pb2 + (Fig. 1b). Because all available evidence indicates that Pb2 + is a full agonist in calmodulin activation, it seems likely that the inhibitory effects of Pb2 + were caused, not by interactions with calmodulin, but by direct interaction between Pb2 + and calcineurin. The fact that Ca2 + is competitive with Pb2 + , fully or partially restoring calcineurin activity both in the presence (Fig. 2) and the absence of calmodulin (Fig. 3), suggests that the Pb2 + -sensitive site can also bind Ca2 + , although with much lower affinity. Ca2 + would not be expected to have significant interactions with the critical cysteine in the A subunit or to replace the active site heavy metals. However, Ca2 + binding to the EF hands of the B subunit enhances calcineurin activity. We therefore tentatively hypothesize that high concentrations of Pb2 + may inhibit calcineurin activity by replacing Ca2 + in the B subunit.
4.2. Possible effects of Pb 2 + on calcineurin acti6ity in 6i6o The intracellular free Pb2 + concentration in chronically Pb2 + -exposed cells is not known. However, Schanne et al. (1989) measured free Ca2 + and free Pb2 + in rat cortical neurons with the indicator 5F-BAPTA, which generates simultaneous, unambiguous NMR peaks for these two ions. Exposing the neurons to 5 mM Pb2 + for up to 2 h resulted in intracellular concentrations of about 30 pM free Pb2 + . Tomsig and Suszkiw (1990) exposed bovine chromaffin cells to 3 mM free Pb2 + for up to 8 min. Using fura-2 and assuming constant [Ca2 + ]i for this time period, they estimated up to 25 pM intracellular free Pb2 + . The concentration of extracellular Pb2 + in both plasma and cerebrospinal fluid is less than 100 nM, even in fairly highly exposed individuals (Cavalleri et al., 1984; Manton and Cook, 1984), but the exposure time is naturally much longer than in these in vitro experiments. Nevertheless, even if we assume that the intracellular free Pb2 + concentration may be as high as 100 pM, Pb2 +
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alone would probably not substantially activate calcineurin in vivo. In the presence of Ca2 + , much lower concentrations of Pb2 + would contribute to calcineurin activation. In intact cells, calmodulin and calcineurin will probably not be activated very much by resting Ca2 + concentrations, with or without Pb2 + in the cytoplasm. However, the intracellular free Ca2 + concentration rises considerably during cellular activity. The global intracellular free Ca2 + concentration may rise to several hundred nM. Localized hot spots of free Ca2 + near the mouths of intracellular release channels or plasma membrane influx channels may have concentrations of dozens of mM (Spigelman et al., 1996), with the Ca2 + concentration rapidly decreasing with distance from the site of Ca2 + entry into the cytoplasm (Smith et al., 1996; Naraghi and Neher, 1997). Activation of many calmodulin-stimulated enzymes will principally occur in a Ca2 + hot spot (Pereschini and Cronk, 1999), and a background of intracellular free Pb2 + will cause an increased intensity of activation in the hot spot and an increased effective volume of the hot spot. Further, hot spots of intracellular Pb2 + may occur at sites of Pb2 + entry. For example, Simons and Pocock (1987) reported that voltage-sensitive calcium channels are even more permeable to Pb2 + than to Ca2 + , which makes it likely that the mouths of calcium channels may be hot spots for both Ca2 + and Pb2 + . If the locations of Ca2 + and Pb2 + hot spots overlap significantly, the degree of calcineurin activation will be a function of the relative and absolute metal concentrations, which will in turn depend on the magnitudes of metal influx and/or release and the effective diffusion distances of the two metals. Therefore, the effects of Pb2 + on calcineurin activity in vivo are probably extremely complex.
Acknowledgements This research was funded by grant ES03572 from the National Institute of Environmental Health Sciences. We would like to thank Drs Teresa Audesirk and Charles Ferguson for dis-
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cussions and suggestions during the course of the research and for critical reading of the manuscript.
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Stimulatory and inhibitory effects of inorganic lead on calcineurin Marcey Kern, Gerald Audesirk * Biology Department, Uni6ersity of Colorado at Den6er, PO Box 173364, Den6er, CO 80217 -3364, USA Received 10 February 2000; accepted 14 June 2000
Abstract Calcineurin is a phosphatase with activity dependent on both Ca2 + /calmodulin binding to the catalytic A subunit and Ca2 + binding to the regulatory B subunit. We have previously shown that Pb2 + activates calmodulin with a threshold of about 100 pM free Pb2 + , and that Pb2 + and Ca2 + are roughly additive in calmodulin activation (Kern et al., NeuroToxicology 21, 353–364 (2000)). In the present study, we evaluated the effects of Pb2 + , with and without Ca2 + and calmodulin, on calcineurin activity. In calmodulin-containing, Ca2 + -free solutions, Pb2 + activated calcineurin with a threshold of about 100 pM free Pb2 + . Maximum calcineurin activity (comparable to that induced by 10 mM Ca2 + ) was reached at about 200 pM free Pb2 + . Higher Pb2 + concentrations reduced activity, although some activity remained even at 2000 pM free Pb2 + . Combined with subsaturating Ca2 + concentrations, as little as 20 pM free Pb2 + enhanced calcineurin activity, but free Pb2 + concentrations greater than 200 pM still reduced activity below maximum. Extremely high Ca2 + concentrations (10 mM) completely reversed the inhibition of activity by 2000 pM free Pb2 + . In the absence of calmodulin, Ca2 + slightly stimulated calcineurin activity. Pb2 + did not substitute for Ca2 + in calmodulin-free activation; in fact, high concentrations of Pb2 + inhibited Ca2 + -mediated activation. We tentatively conclude that low concentrations of free Pb2 + activate calcineurin by activating calmodulin. Higher concentrations reduce calcineurin activity, perhaps by binding to the B subunit. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calcineurin; Protein phosphatase 2B; Lead; Pb2 + , calcium; Ca2 +
1. Introduction Calcineurin (protein phosphatase 2B) is a serine/threonine protein phosphatase with wide* Corresponding author. Tel.: +1-303-5562593; fax: +1303-5564352. E-mail address: [email protected] (G. Audesirk).
spread distribution in the brain, immune system, and other tissues. The phosphatase activity of calcineurin is dependent on both Ca2 + /calmodulin binding to the catalytic A subunit and Ca2 + binding to the regulatory B subunit (Stewart et al., 1982; Klee et al., 1988; Stemmer and Klee, 1994; Perrino et al., 1995; Perrino and Soderling, 1998). Inorganic lead (Pb2 + ) binds to many Ca2 + -binding proteins, where it may act as an agonist,
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 2 5 8 - 4
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antagonist, or partial agonist. For example, Pb2 + acts as an agonist in activating calmodulin (Goldstein and Ar, 1983; Habermann et al., 1983; Kern et al., 2000), as an antagonist in inhibiting Ca2 + -dependent inactivation of voltage-sensitive calcium channels (Sun and Suszkiw, 1995), and as a partial agonist in activating Ca2 + -dependent isoforms of protein kinase C (Long et al., 1994; Tomsig and Suszkiw, 1995; but see also Saijoh et al., 1988; Murakami et al., 1993). We have previously shown that Pb2 + is a full agonist in activation of calmodulin, as assayed by calmodulin-dependent stimulation of cyclic nucleotide phosphodiesterase (PDE) and stimulation of the fluorescence of hydrophobic probes (Kern et al., 2000). The threshold for calmodulin activation in both assays is about 100 pM free Pb2 + . Probably of more physiological importance, Pb2 + and Ca2 + are roughly additive in activating calmodulin-dependent stimulation of PDE. Therefore, we hypothesized that Pb2 + would act as an agonist for calmodulin-dependent stimulation of calcineurin activity, through Pb2 + / calmodulin binding to the catalytic A subunit. Further, we predicted that Pb2 + and Ca2 + would be roughly additive in calmodulin-dependent stimulation of calcineurin activity. The possible effects of Pb2 + binding to the EF hands of the B subunit (Kakalis et al., 1995) could not be predicted. Further, calcineurin apparently has at least one cysteine in the A subunit that affects enzymatic activity (King, 1986). Because Pb2 + has a high affinity for cysteine residues (Al-Modhefer et al., 1991), Pb2 + might also alter calcineurin activity by binding to this cysteine residue. Overall, then, the effects of Pb2 + on calcineurin activity could range from simple activation to complex, possibly concentration-dependent stimulatory and inhibitory actions. We evaluated the effects of Pb2 + , with and without Ca2 + and calmodulin, on calcineurin activity. In the presence of calmodulin, low concentrations of free Pb2 + ( 5200 pM) stimulated calcineurin activity, but higher Pb2 + concentrations reduced activity. Stimulation of calcineurin activity by combinations of Ca2 + and low Pb2 + concentrations was roughly additive. High Ca2 + concentrations reversed the reduction in activity
caused by high Pb2 + concentrations. Ca2 + , but not Pb2 + , supported a low level of calcineurin activity in the absence of calmodulin.
2. Methods
2.1. Materials Calcineurin was purchased from Boehringer Mannheim, calmodulin from Calbiochem, EGTA from J.T. Baker, and a phosphorylated peptide corresponding to a portion of the RII subunit of cyclic AMP-dependent protein kinase (DonellaDeana et al., 1994) from Biomol. Calcineurin phosphatase activity was measured as phosphate released from the RII subunit. Phosphate was measured by its reaction with molybdate/malachite green (Lanzetta et al., 1979), using the reagents of the serine/threonine phosphatase assay kit from Promega. All other chemicals were purchased from Sigma.
2.2. Assay methods The reaction mixture (100 ml final volume) consisted of 100 nM calcineurin, 750 nM calmodulin, 25 mM phosphorylated RII peptide, 2% trehalose, 1 mM EGTA, 10 mM MgCl2, and 25 mM Tris (pH 7.3), with CaCl2 or Pb(NO3)2 added to produce solutions with variable concentrations of free Ca2 + and/or free Pb2 + ions. For experiments in which high concentrations of Ca2 + and Pb2 + were needed (see Fig. 3), the buffer also contained 1 mM nitrilotriacetic acid (NTA). All solutions were checked daily for phosphate contamination. Free metal concentrations were estimated using the Chelator program (Schoenmakers et al., 1992) and the stability constants for ligand/metal complexes given in Martell and Smith (1974). The stability constants used for EGTA/metal complexes (all given for 20°C and 0.1 M ionic strength) were (1) Pb2 + /EGTA: 14.71; (2) Ca2 + / EGTA: 10.97; and (3) Mg2 + /EGTA: 5.21. The stability constants for NTA/metal complexes were (1) Pb2 + /NTA: 11.39; (2) Ca2 + /NTA: 6.41; and (3) Mg2 + /NTA: 5.41. The Chelator program corrects the stability constants for other temperatures
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and ionic strengths. Other stability constants (e.g. for Pb2 + /EGTA: 13.48 (Simons 1995)) give slightly higher free metal ion concentrations than those given in the Results, but do not affect the interpretation of the experiments. To measure the phosphatase activity of calcineurin, all of the ingredients except the RII peptide were mixed together and the reaction was started by rapid addition of the RII peptide. The
Fig. 1. Calcineurin activity as a function of free Ca2 + and/or Pb2 + ion concentrations. All points in all figures represent mean9 s.e.m. of at least six replicates in at least three different experiments. (a) Calcineurin activity in the presence of Ca2 + alone (filled circles) or variable Ca2 + plus 100 pM free Pb2 + (open circles). The curves are four-parameter Hill equations fit to the data points. The EC50 for activation by Ca2 + alone was approximately 740 950 nM; in the presence of 100 pM free Pb2 + , the EC50 for activation by Ca2 + was reduced to approximately 340 930 nM. (b) Calcineurin activity in the presence of Pb2 + alone (filled circles) or variable Pb2 + plus 500 nM free Ca2 + (open circles). Between 20 and 100 pM free Pb2 + , the combination of Pb2 + and Ca2 + stimulated significantly greater calcineurin activity than was stimulated by either cation alone. Between 200 and 2000 pM free Pb2 + , calcineurin activity was not affected by 500 nM Ca2 + .
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reaction mixture was incubated at 30°C for 30 min, then the reaction was stopped by adding 100 ml of the Promega molybdate/malachite green dye mixture. The resulting solution was incubated at room temperature for 20 min, after which the absorbance was measured at 600 nm. Preliminary experiments showed that this incubation period was within the linear range of both calcineurin activity (at this substrate concentration) and the molybdate/malachite green phosphate measurement. Calcineurin activity in the absence of calmodulin was measured with the same protocol but with calmodulin omitted and the incubation period increased to 2 h. All data points shown in the figures represent means 9s.e.m. of at least six replicates in at least three separate experiments. All calcineurin activity measurements given in the Results were corrected by subtracting the absorbance of reaction mixtures containing RII peptide but with calcineurin omitted. Ca2 + - and Pb2 + -independent activity (in the presence of calmodulin) varied in different experiments from about 3 to 15% of the activity in the presence of saturating calmodulin and Ca2 + . Phosphate release was estimated by comparison with standards of known phosphate concentration. Maximum calcineurin activity in the presence of excess calmodulin and saturating free Ca2 + was : 2.59 0.3 (mean 9 s.e.m.) picomoles of phosphate released per minute per picomole calcineurin. This is equivalent to about 31 pm/ min/mg, which is reasonably consistent with the 57 pm/min/mg determined by Donella-Deana et al. (1994) using the same substrate but a different buffer and pH 8. For ease of comparison, calcineurin activity in the Results is presented as a fraction of the activity in solutions containing saturating calmodulin and free Ca2 + (10 mM or 1 mM; see Fig. 1).
2.3. Statistical analysis Activation curves of calcineurin activity as a function of free cation concentration were generated by fitting the data to a four-parameter Hill plot (Sigma Plot, SPSS). The curve fits were then used to estimate thresholds and EC50 values. Where appropriate, data were also analyzed by
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ANOVA, followed by Dunnett’s test for multiple comparisons between a single control and several experimental conditions, or by the Student – Neumann–Keuls test for multiple comparisons among treatments. Effects were considered significant at the 0.05 level.
3. Results
3.1. Acti6ation of calcineurin by Ca 2 + and Pb 2 + Calcineurin activity was measured in nominally Ca2 + -free conditions (1 mM EGTA, no added Ca2 + ) and in solutions containing approximate free Ca2 + concentrations of 50 nM to 1 mM (filled circles in Fig. 1a). Ca2 + -dependent activity was statistically significant at 500 nM free Ca2 + and saturated at 2– 10 mM free Ca2 + . The EC50 for calcineurin activation was :740 9 50 nM free Ca2 + (mean9s.e.m.). This EC50 concentration is similar to that reported in the literature (Stemmer and Klee, 1994). Calcineurin activity was similarly measured in nominally Pb2 + -free conditions (1 mM EGTA, no added Pb2 + ) and in solutions containing approximate free Pb2 + concentrations of 10 – 2000 pM (filled circles in Fig. 1b). Pb2 + activated calcineurin activity at concentrations of 100 pM and above. Calcineurin activity peaked at about 200 pM free Pb2 + at a level that was not significantly different from the activity in the presence of saturating free Ca2 + (P \ 0.5). Free Pb2 + concentrations above 200 pM reduced calcineurin activity, but some phosphatase activity remained even at 2000 pM free Pb2 + .
3.2. Interactions between Ca 2 + and Pb 2 + in calcineurin acti6ation Pb2 + and Ca2 + are roughly additive in activation of calmodulin (Kern et al., 2000). Therefore, we predicted that Pb2 + and Ca2 + should act additively in activation of calcineurin. We tested the effects of suboptimal concentrations of free Pb2 + in the presence of variable free Ca2 + , and vice versa.
First, calcineurin activity was measured in solutions containing a constant 100 pM free Pb2 + and concentrations of free Ca2 + varying between 100 nM and 10 mM. This concentration of free Pb2 + was chosen because it supports low calcineurin activity (Fig. 1b), and therefore would enable us to detect positive or negative interactions between Ca2 + and Pb2 + . One hundred pM free Pb2 + augmented calcineurin activation by Ca2 + , lowering the free Ca2 + threshold and shifting Ca2 + activation curve to the left; i.e. lower concentrations of free Ca2 + were needed to activate calcineurin in the presence of Pb2 + (open circles in Fig. 1a). The EC50 for activation by Ca2 + in the presence of 100 pM free Pb2 + was : 340+ 30 nM free Ca2 + , compared to 740 nM free Ca2 + in the absence of Pb2 + . The maximum calcineurin activity remained the same at high free Ca2 + concentrations with or without 100 pM free Pb2+. Second, we tested the effects of variable free Pb2 + concentrations on the activation of calcineurin by 500 nM free Ca2 + , a Ca2 + concentration that supports modest calcineurin activity (see Fig. 1a). Low concentrations of free Pb2 + (i.e. between 20 and 100 pM) increased calcineurin activity compared to that caused by 500 nM free Ca2 + alone (open circles in Fig. 1b). Maximum calcineurin activity still occurred with about 200 pM free Pb2 + , and this activity was not significantly different from that caused by saturating free Ca2 + (P\0.5). The reduction of calcineurin activity caused by high concentrations of free Pb2 + (over 200 pM) was not affected by 500 nM free Ca2 + (Fig. 1b). This might indicate that the reduction is caused by binding of Pb2 + to a site on calcineurin that does not bind Ca2 + . However, Pb2 + has much higher affinity than Ca2 + for many Ca2 + -binding proteins (e.g. Markovac and Goldstein, 1988; Long et al., 1994; Tomsig and Suszkiw, 1995; Kern et al., 2000). Therefore, it is also possible that Pb2 + at concentrations above 200 pM may simply outcompete 500 nM free Ca2 + for a site on calcineurin for which Ca2 + is either permissive or stimulatory, and inhibit enzyme activity. If so, then very high free Ca2 + concentrations should be able to reverse the reduction of activity caused
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not support calmodulin-independent calcineurin activity at any concentration from 100 pM to 50 nM (data not shown). Further, Pb2 + reduced (2 nM) or eliminated (50 nM) the stimulation of calmodulin-independent activity by 10 mM Ca2 + (Fig. 3). Finally, 10 mM Ca2 + supported full calmodulin-dependent activity in the presence of 2 nM Pb2 + (Fig. 2) but did not support full calmodulinindependent activity in the presence of 2 nM Pb2 + (Fig. 3). This apparent discrepancy probably results from the fact that calmodulin-dependent calcineurin activity was over 30-fold greater than calmodulin-independent activity. Therefore, in the presence of calmodulin (Fig. 2), a small inhibition of activity caused by calmodulin-independent effects of Pb2 + could not be detected against the high background of calmodulin-dependent activity stimulated by Ca2 + and Pb2 + . Fig. 2. The reduced calcineurin activity caused by 2 nM free Pb2 + was completely reversed by 10 mM free Ca2 + . All data points, including 10 mM free Ca2 + and 2 nM free Pb2 + , represent new experiments (i.e. the data points are not repeated from earlier figures). Asterisk: significantly different from activity with 10 mM Ca2 + .
by Pb2 + . To test this hypothesis, we compared calcineurin activity in the presence of 10 mM free Ca2 + alone (a saturating concentration; see Fig. 1a), 2 nM free Pb2 + alone, or both in combination (Fig. 2). We found that 10 mM free Ca2 + reversed the activity reduction caused by 2 nM free Pb2 + .
3.3. Effects of Ca 2 + and Pb 2 + on calmodulin-independent calcineurin acti6ity Calcineurin has limited activity in the absence of calmodulin (Haddy et al., 1992; Perrino et al., 1995). We examined the effects of Ca2 + and Pb2 + , alone and in combination, on this calmodulin-independent activity. In agreement with many reports in the literature, we found that significant calmodulin-free calcineurin activity required Ca2 + (Fig. 3). Calmodulin-independent activity in the presence of 10 mM free Ca2 + was about 3% of the activity that occurred with supersaturating calmodulin concentrations. Pb2 + did
Fig. 3. The effects of Ca2 + and Pb2 + on calmodulin-independent calcineurin activity. Phosphatase activity was not significantly different from zero in the absence of either cation (black bar). Saturating free Ca2 + (10mM) in calmodulin-free solutions (open bar) stimulated about 3% of the maximum activity seen in solutions containing both saturating calmodulin and Ca2 + . Pb2 + reduced the Ca2 + -dependent, calmodulin-independent activity (striped bars). Asterisks: significantly different from activity with 10 mM Ca2 + .
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4. Discussion
4.1. Stimulatory and inhibitory effects of Pb 2 + on calcineurin acti6ity The activity of calcineurin is controlled by a complex interaction among several factors, including calmodulin binding to the A (catalytic) subunit, the binding of Ca2 + to calmodulin and independently to the B subunit (Klee et al., 1988; Stemmer and Klee, 1994; Perrino et al., 1995; Perrino and Soderling, 1998), the presence and oxidation state of iron and zinc in the active site (King and Huang, 1986; Kissinger et al., 1995; Wang et al., 1996), and the oxidation state of a critical cysteine in the A subunit (King, 1986). We found that Pb2 + had both stimulatory and inhibitory effects on calcineurin activity (Fig. 1b). In principle, these effects might be caused by interactions of Pb2 + with calmodulin or with any of several sites on calcineurin itself. We had previously shown that Pb2 + activated calmodulin, with a threshold of about 100 – 300 pM free Pb2 + (Kern et al., 2000). High free Pb2 + concentrations (from about 1000 pM up to 5000 pM, the highest concentration tested) activated calmodulin to the same extent as saturating Ca2 + . Further, Pb2 + and Ca2 + were additive in calmodulin activation. Finally, there were no inhibitory effects of any concentration of Pb2 + , with or without Ca2 + . Therefore, Pb2 + appears to be a full agonist for calmodulin activation. In the present study, low concentrations of Pb2 + stimulated calcineurin activity (Fig. 1b), the maximum activity obtained with Pb2 + alone (at 200 pM) did not differ from the maximum activity obtained with saturating concentrations of Ca2 + , and low concentrations of Pb2 + were roughly additive with Ca2 + in activating calcineurin, all of which would be expected of Pb2 + activation of calmodulin. No concentration of Pb2 + from 100 pM through 50 nM was able to support significant calmodulin-independent activity, which would be expected if Pb2 + stimulated calcineurin activity by a direct interaction between Pb2 + and calcineurin itself. Therefore, we conclude that Pb2 + stimulates calcineurin by activation of calmodulin.
There was progressively less calcineurin activation with free Pb2 + concentrations above 200 pM, although there was still some stimulation of calcineurin activity even at 2000 pM free Pb2 + (Fig. 1b). Because all available evidence indicates that Pb2 + is a full agonist in calmodulin activation, it seems likely that the inhibitory effects of Pb2 + were caused, not by interactions with calmodulin, but by direct interaction between Pb2 + and calcineurin. The fact that Ca2 + is competitive with Pb2 + , fully or partially restoring calcineurin activity both in the presence (Fig. 2) and the absence of calmodulin (Fig. 3), suggests that the Pb2 + -sensitive site can also bind Ca2 + , although with much lower affinity. Ca2 + would not be expected to have significant interactions with the critical cysteine in the A subunit or to replace the active site heavy metals. However, Ca2 + binding to the EF hands of the B subunit enhances calcineurin activity. We therefore tentatively hypothesize that high concentrations of Pb2 + may inhibit calcineurin activity by replacing Ca2 + in the B subunit.
4.2. Possible effects of Pb 2 + on calcineurin acti6ity in 6i6o The intracellular free Pb2 + concentration in chronically Pb2 + -exposed cells is not known. However, Schanne et al. (1989) measured free Ca2 + and free Pb2 + in rat cortical neurons with the indicator 5F-BAPTA, which generates simultaneous, unambiguous NMR peaks for these two ions. Exposing the neurons to 5 mM Pb2 + for up to 2 h resulted in intracellular concentrations of about 30 pM free Pb2 + . Tomsig and Suszkiw (1990) exposed bovine chromaffin cells to 3 mM free Pb2 + for up to 8 min. Using fura-2 and assuming constant [Ca2 + ]i for this time period, they estimated up to 25 pM intracellular free Pb2 + . The concentration of extracellular Pb2 + in both plasma and cerebrospinal fluid is less than 100 nM, even in fairly highly exposed individuals (Cavalleri et al., 1984; Manton and Cook, 1984), but the exposure time is naturally much longer than in these in vitro experiments. Nevertheless, even if we assume that the intracellular free Pb2 + concentration may be as high as 100 pM, Pb2 +
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alone would probably not substantially activate calcineurin in vivo. In the presence of Ca2 + , much lower concentrations of Pb2 + would contribute to calcineurin activation. In intact cells, calmodulin and calcineurin will probably not be activated very much by resting Ca2 + concentrations, with or without Pb2 + in the cytoplasm. However, the intracellular free Ca2 + concentration rises considerably during cellular activity. The global intracellular free Ca2 + concentration may rise to several hundred nM. Localized hot spots of free Ca2 + near the mouths of intracellular release channels or plasma membrane influx channels may have concentrations of dozens of mM (Spigelman et al., 1996), with the Ca2 + concentration rapidly decreasing with distance from the site of Ca2 + entry into the cytoplasm (Smith et al., 1996; Naraghi and Neher, 1997). Activation of many calmodulin-stimulated enzymes will principally occur in a Ca2 + hot spot (Pereschini and Cronk, 1999), and a background of intracellular free Pb2 + will cause an increased intensity of activation in the hot spot and an increased effective volume of the hot spot. Further, hot spots of intracellular Pb2 + may occur at sites of Pb2 + entry. For example, Simons and Pocock (1987) reported that voltage-sensitive calcium channels are even more permeable to Pb2 + than to Ca2 + , which makes it likely that the mouths of calcium channels may be hot spots for both Ca2 + and Pb2 + . If the locations of Ca2 + and Pb2 + hot spots overlap significantly, the degree of calcineurin activation will be a function of the relative and absolute metal concentrations, which will in turn depend on the magnitudes of metal influx and/or release and the effective diffusion distances of the two metals. Therefore, the effects of Pb2 + on calcineurin activity in vivo are probably extremely complex.
Acknowledgements This research was funded by grant ES03572 from the National Institute of Environmental Health Sciences. We would like to thank Drs Teresa Audesirk and Charles Ferguson for dis-
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cussions and suggestions during the course of the research and for critical reading of the manuscript.
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