The Calmodulin Regulator Protein, PEP-19, Sensitizes ATP-induced Ca²⁺ Release

Background: PEP-19 modulates the kinetics of Ca²⁺ binding to CaM.

Results: An acidic region in PEP-19 binds Ca²⁺ and is essential for both modulating Ca²⁺ binding to CaM and sensitizing cells to ATP-induced Ca²⁺ release.

Conclusion: Simply binding to CaM is not sufficient to account for the biological activities of PEP-19.

Significance: Regulating ligand-induced Ca²⁺ release gives PEP-19 the potential to broadly affect cell signaling.

Abstract

PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca²⁺ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca²⁺ binding activity, which may modulate Ca²⁺ binding to CaM by stabilizing an initial Ca²⁺-CaM complex or by electrostatically steering Ca²⁺ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca²⁺ dynamics, we tested the hypothesis that it influences ligand-dependent Ca²⁺ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca²⁺ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca²⁺ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca²⁺ binding to CaM greatly inhibit its effect on ATP-induced Ca²⁺ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca²⁺ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.

Introduction

PEP-19 (Purkinje cell protein 4, Pcp4) is a small protein (62 amino acids) with no known intrinsic activity other than binding to CaM² in the presence or absence of Ca²⁺. Although it was originally identified in the central nervous system, PEP-19 mRNA is also found in human bladder, kidney, prostate, uterus, thyroid, and adrenal tissues (1). Changes in expression levels suggest biological roles for PEP-19 in both normal and pathological conditions. For example, PEP-19 mRNA levels are significantly reduced in a mouse model for Parkinson disease (2) and in the prefrontal cortex of alcoholics (3), but its levels are increased in anergic B cells (4) and in human uterine leiomyomas (5).

Animal and cellular model systems have demonstrated effects of PEP-19 on diverse cellular processes. PEP-19 null mice show a dramatic reduction in long term plasticity at synapses between granule cell parallel fibers and Purkinje cells (6). Overexpression of PEP-19 in PC12 cells increases neurite outgrowth (7), and premature neuronal differentiation is seen in transgenic mice with three copies of the PEP-19 gene (Pcp4) (8). The latter suggests a role for PEP-19 in Down syndrome because the human PEP-19 gene (PCP4) is present on chromosome 21. In addition, PEP-19 has anti-apoptotic activity when expressed in PC12 and HEK293T cells (9, 10), and it provides protection against Ca²⁺ overload in cortical neurons (10). These experimental observations are consistent with a proposed neuroprotective role for PEP-19 based on expression patterns in neuronal tissues that are susceptible to Huntington and Alzheimer diseases (11).

The above studies emphasize the need to understand the mechanism of action of PEP-19. Two models for PEP-19 have been proposed based on studies using peptides and the homologous proteins neurogranin (Ng) and neuromodulin (12–14). The first, or camstatin model, proposes that PEP-19 competitively inhibits activation of CaM target proteins. The second, or calpacitin model, proposes that PEP-19 binds with high affinity to apo-CaM to retard its release from PEP-19, thereby affecting the temporal profile of available CaM during a Ca²⁺ pulse. We proposed an alternative or additional mechanism for PEP-19 based on its ability to modulate the Ca²⁺ binding properties of CaM. Specifically, PEP-19 increases both the Ca²⁺ k_on and k_off rates at the C-domain of CaM up to 40-fold with little effect on the K_Ca (15). We also showed that an acidic sequence located adjacent to the IQ motif is required to modulate Ca²⁺ binding to the C-domain of CaM, even though it has no apparent intrinsic affinity for CaM (16). Thus, the acidic/IQ motif of PEP-19 has the potential to modulate the rate-limiting kinetics of Ca²⁺ binding to CaM.

This study investigates the molecular mechanism by which PEP-19 modulates Ca²⁺ binding to CaM, and it tests the hypothesis that the biological activities of PEP-19 rely on synergy between the biochemical properties of its acidic and IQ sequences. Our results show that the acidic sequence in PEP-19 has intrinsic metal binding properties that play a role in increasing the rates of Ca²⁺ binding to CaM, at least in part, by electrostatically steering Ca²⁺ to and from Ca²⁺ binding sites III and/or IV. We also show that PEP-19 sensitizes HeLa cells to ATP-dependent Ca²⁺ release and that this effect is greatly reduced or eliminated by mutations in PEP-19 that inhibit or eliminate its ability to modulate Ca²⁺ binding to CaM. Tuning the activities of Ca²⁺ mobilization pathways by PEP-19 greatly expands the biological significance of this small regulator of CaM signaling.

EXPERIMENTAL PROCEDURES

Mutagenesis and Protein Purification

QuikChange II XL site-directed mutagenesis kit (Stratagene) was used to generate a panel of PEP-19 mutants. CaM and C-CaM (isolated C domain of calmodulin) were decalcified by addition of 5 mm EDTA and 0.1 mm BAPTA as a UV marker and then desalting on a Bio-Gel P2 column (Bio-Rad) in 10 mm NH₄HCO₃ that had been decalcified using a Ca²⁺ sponge column (Molecular Probes). Decalcified proteins were then lyophilized and resuspended in desired buffers. Protein concentrations were estimated using an extinction coefficient of ϵ_{276 nm} = 0.18 ml⁻¹ mg⁻¹ for C-CaM and ϵ_{215 nm} = 0.59 ml⁻¹ mg⁻¹ for PEP-19.

Ca²⁺ Binding Measurements

The rate of Ca²⁺ dissociation (k_off) from CaM or C-CaM in the presence or absence of PEP-19 derivatives was determined using stopped-flow fluorescence and the Ca²⁺-sensitive dye Quin-2 as described previously (15). Typically, solutions of 2–5 μm CaM or C-CaM in 20 mm MOPS, pH 7.5, 100 mm KCl, 30 μm CaCl₂ were rapidly mixed with 20 mm MOPS, pH 7.5, 300 μm Quin-2. Excess free Ca²⁺ and Ca²⁺ that is rapidly released from the N-domain of CaM bind to Quin-2 in the 1.7-ms dead time of the stopped-flow instrument. The subsequent increase in Quin-2 fluorescence is due to binding Ca²⁺ released slowly from the C-domain. Experiments were performed at 23 °C using an Applied Photophysics Ltd. (Leatherhead, UK) model SX20 MV sequential stopped-flow spectrofluorimeter with a 150 watt Xe/Hg lamp.

Equilibrium Ca²⁺ binding constants for CaM in the presence or absence of PEP-19 derivatives were determined using tyrosine fluorescence at 23 °C as described previously (17). Data were collected with a QuantaMaster fluorimeter (Photo Technology International). Intrinsic Tyr emission spectra were recorded from 290 to 320 nm with the excitation wavelength of 276 nm. Solutions contained 20 mm MOPS, pH 7.5, 0 or 100 mm KCl, 1 mm EGTA, 1 mm HEDTA, 1 mm nitrilo-2,2′,2′-triacetic acid, 5 μm CaM or C-CaM with or without PEP-19 or its derivatives. Calcium was added from a concentrated stock prepared in the same buffer with CaM, PEP-19, and chelators, so that only the concentration of Ca²⁺ changes during the titration even though the volume increases. The concentration of total Ca²⁺ needed to achieve a desired free Ca²⁺ concentration was determined using the on-line calculator MaxChelator. Control titrations were performed using Br₂BAPTA as an indicator instead of CaM or C-CaM to confirm that the calculated free Ca²⁺ was accurate at high and low ionic strength. The K_Ca for Br₂BAPTA is 1.59 μm at 100 mm KCl and 0.15 μm at 10 mm KCl (18).

Tyrosine fluorescence intensity was plotted against the free Ca²⁺ concentration and fit to the following form of the Hill Equation 1,

where [Ca²⁺] is the free Ca²⁺ concentration; F is the fluorescence intensity at a given free Ca²⁺ concentration; F_min is the initial fluorescence intensity in the absence of added Ca²⁺; F_max is the fluorescence at maximal Ca²⁺; K_Ca is the concentration of Ca²⁺ at which the change in fluorescence is half-maximal, and n is the Hill coefficient.

NMR Methodology

NMR experiments were performed on a Bruker DRX 600 MHz spectrometer equipped with a 5-mm triple resonance cryoprobe at 298 K. Protein samples were dissolved in buffer containing 10 mm imidazole, 5% D₂O (v/v), pH 6.3, 100 mm KCl. ¹H,¹⁵N HSQC spectra were used to determine residues in PEP-19 that are affected by binding to C-CaM. Briefly, ¹H,¹⁵N HSQC spectra were collected during titration of ¹⁵N-labeled PEP-19 with C-CaM in the presence or absence of Ca²⁺. Characteristics of fast exchange were seen at saturating Ca²⁺, so backbone amides could be assigned by following cross-peaks during the titration. Slow exchange was seen in the apo-state, so assignments in the bound state were made using HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, ¹⁵N HSQC-TOCSY, and ¹⁵N-edited NOESY-HSQC experiments. All NMR spectra were processed and analyzed using Topspin 2.0 (Bruker) and FELIX 2004 (MSI, San Diego). ¹H chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate, and ¹⁵N/¹³C chemical shifts were referenced indirectly using their respective gyromagnetic ratios. The average amide chemical shift change was calculated using Equation 2,

where ΔδH is the change in ¹H chemical shift and ΔδN is the change in ¹⁵N chemical shift.

Calcium Imaging

Calcium imaging was performed exactly as described previously (19). HeLa cells were transfected with yellow fluorescent protein (YFP) only (control) or co-transfected with YFP and PEP-19 constructs at a DNA ratio of 1:4 using Lipofectamine 2000. Twenty four hours after transfection, single cell calcium responses evoked by NaATP were recorded from all YFP-positive cells in a given field. All experiments were repeated at least three times, and the data were pooled for statistical analysis. The actual number of single cell records averaged for each condition is indicated above the bars in Fig. 6c.

FIGURE 6.

Effect of PEP-19 and its derivatives on ATP-induced intracellular Ca²⁺ release. Western blots in a show the relative level of expression of Myc-tagged PEP-19 and mutant proteins in HeLa cells that were transiently transfected with the corresponding expression plasmids. b shows intracellular Ca²⁺ levels in single cells in response to increasing concentrations of ATP. c shows the percentage of cells from each group that showed increased intracellular Ca²⁺ in response to 0.1 μm ATP. d and e show the effect of PEP-19 and mutated derivatives on peak Ca²⁺ levels and Ca²⁺ oscillation frequency, respectively, in response to 1 μm and 10 μm ATP. Data in d and e are represented as mean ± S.E. Statistical significance was determined with a Student's t test with * indicating p < 0.05 versus YFP and ** indicating p < 0.05 versus PEP-19.

RESULTS

PEP-19 Proteins Generated for Study

We used amide chemical shift perturbation to identify residues in PEP-19 that experience significant structural transitions upon binding to the C-domain of CaM because these residues will likely play key roles in regulating Ca²⁺ binding to CaM. C-CaM, which encodes residues 76–148 of CaM, was used for these experiments because we showed previously that PEP-19 binds to C-CaM and had the same effects on its Ca²⁺-binding proteins as seen for full-length CaM (20). Fig. 1, a and b, shows that backbone amide chemical shifts for residues 1–30 in PEP-19 are unchanged upon binding to C-CaM in the absence or presence of Ca²⁺. Because free PEP-19 is intrinsically disordered (21), these data show that residues 1–30 remain disordered when bound to C-CaM. Amide chemical shift perturbations are restricted to residues in the acidic/IQ region of PEP-19 upon binding to either apo- or Ca²⁺-C-CaM.

FIGURE 1.

Amide chemical shift changes in PEP-19 upon binding to C-CaM are restricted to the acidic/IQ region. Average amide chemical shift perturbations were determined for residues in ¹⁵N-labeled PEP-19 caused by binding to either apo (a) or Ca²⁺-bound (b) C-CaM as described under “Experimental Procedures.” The arrowheads indicate acidic residues that could potentially bind Ca²⁺. The asterisk indicates Pro-37, which does not have a backbone amide. The core IQ sequence shown in c is based on comparison of multiple IQ motif proteins. Residues denoted by X, Y, Z, and −Y, −X, and −Z in native PEP-19 correspond to potential Ca²⁺ ligand positions based on a canonical EF-hand Ca²⁺-binding motif. PEPΔAc was generated by deletion of residues Glu-28 to Arg-41. The open arrowheads indicate that Val-26 is shifted to the position of Glu-40 in PEPΔAc. Residues 28–40 were randomly scrambled in PEPscram while retaining Pro-37. Black arrowheads indicate positions of point mutations.

Based on the above chemical shift perturbations, two sets of proteins were generated to test the biochemical and functional significance of the acidic sequence in PEP-19 (see Fig. 1c). The acidic sequence is deleted in PEPΔAc such that Val-26 effectively substitutes for Glu-40 of native PEP-19. We anticipated that a hydrophobic residue at this position would promote association of PEPΔAc with both the N- and C-domains of CaM because a Phe residue at the homologous position in the Ca_V1.2 channel anchors its IQ region to the N-domain (22). Residues in the acidic sequence of PEPscram are randomized to determine whether the native sequence is important for modulating Ca²⁺ binding to CaM or whether a cluster of negative charges is sufficient.

The second set of proteins was designed to test the functional significance of sequence similarity between the acidic region of PEP-19 and the consensus EF-hand Ca²⁺-binding site where alternating residues provide oxygens to coordinate Ca²⁺ at X, Y, Z and −Y,−X, and −Z positions (see Fig. 1c). Thus, Ala was substituted individually for Glu-29, Asp-31, Asp-33, Asp-35, or Glu-40. In addition, Pro-37 was changed to Gly to test the hypothesis that backbone constraints imposed by the cyclized Pro side chain dictates the relative positions of adjacent acidic residues when PEP-19 is bound to CaM, thereby affecting Ca²⁺ binding.

Deletion of the Acidic Sequence Prevents Modulation of Ca²⁺ Binding to CaM

Calcium-dependent Tyr fluorescence was used to measure the K_Ca of the C-domain of CaM in the presence or absence of native and mutated PEP-19. Table 1 shows that neither native PEP-19 nor its mutated derivatives have large effects on K_Ca, although most decreased the cooperativity of Ca²⁺ binding.

TABLE 1.

Effect of mutants on Ca²⁺ binding to CaM

Apparent K_Ca values were determined by monitoring Tyr fluorescence during titration with Ca²⁺ and fitting the data to the Hill equation as described under “Experimental Procedures.” ND indicates not determined. Calcium dissociation rates (k_off) were derived by fitting Ca²⁺ dissociation curves to mono- or diexponential equations. Values for k_off are the average of 3–5 determinations in the presence or absence of 20 μm PEP-19 proteins. Stoichiometry of Ca²⁺ release was determined by calibrating Quin-2 fluorescence. Rates of Ca²⁺ association (k_on) were calculated from k_on = k_off/K_Ca.

PEP-19 protein	Equilibrium Ca2+ binding		Binding kinetics			Stoichiometry
	KCa	Hill coefficient	koff, 1	koff, 2	kon	Ca2+/protein
	μm		s−1	s−1	s−1 μm−1
None	1.6 ± 0.1	1.8 ± 0.1	10.4 ± 0.2		6.5	2.0
PEP-19	2.0 ± 0.1	1.1 ± 0.01	298 ± 10		149	1.8
mycPEP-19	2.0 ± 0.2	1.3 ± 0.04	303 ± 15		151	1.8
PEPΔAc	ND	ND	11.7 ± 0.6	36 ± 4	ND	1.7
PEPscram	1.9 ± 0.2	1.6 ± 0.05	10.6 ± 0.4		5.7	2.0
PEP(E29A)	2.3 ± 0.1	1.1 ± 0.03	276 ± 11		120	1.8
PEP(D31A)	2.4 ± 0.1	1.1 ± 0.01	157 ± 5		65	1.7
PEP(D33A)	1.6 ± 0.1	1.4 ± 0.04	94 ± 6		59	1.8
PEP(D35A)	2.4 ± 0.1	1.1 ± 0.1	288 ± 12		120	1.8
PEP(P37G)	1.6 ± 0.2	1.5 ± 0.02	69 ± 5		43	1.9
PEP(E40A)	2.4 ± 0.2	1.1 ± 0.03	159 ± 8		66	1.7

The relatively slow Ca²⁺ k_off rate of 10.4 s⁻¹ for free CaM in Table 1 is due to dissociation of 2 Ca²⁺ from the C-domain because dissociation of Ca²⁺ from the N-domain is very rapid and occurs in the dead-time (1.7 ms) of the stopped-flow fluorimeter. PEP-19 greatly increases the rate of Ca²⁺ dissociation to about 300 s⁻¹, but the stoichiometry remains 2 Ca²⁺ released per CaM. Table 1 shows that deletion of the acidic sequence in PEPΔAc prevents the increase in Ca²⁺ k_off. Thus, the acidic sequence of PEP-19 is required for modulation of Ca²⁺ binding to CaM.

Interestingly, the stoichiometry of Ca²⁺ release in the presence of PEPΔAc is 4 Ca²⁺/mol of CaM instead of 2 seen the presence of all other PEP-19 proteins. This is consistent with the above prediction that PEPΔAc binds to both the N- and C-domains of CaM, thereby slowing the rate of release of Ca²⁺ from the N-domain as is seen for other CaM-binding proteins and peptides (23). We confirmed this mode of binding using a donor- and acceptor-labeled CaM (CaM(DA)) (24), which gives a large decrease in fluorescence due to FRET when CaM adopts a compact structure upon binding both domains to one peptide. Fig. 2 shows that fluorescence from CaM(DA) is not greatly affected by native PEP-19 because it binds preferentially to the C-domain of CaM, but a large decrease in fluorescence is seen upon binding to either PEPΔAc or a CaM-binding peptide from CaM kinase II, CKII(293–312), which is known to bind to both domains of CaM (25). As a further test, we generated PEP(E40F), with Phe at the homologous position to the Phe that anchors the IQ motif of the Ca_V1.2 channel to the N-domain of CaM (22). Fig. 2 shows that PEP(E40F) also causes a large decrease in fluorescence upon binding to CaM(DA). These results show that the absence of an appropriately positioned hydrophobic group in the acidic region of PEP-19 allows preferential binding to the C-domain of CaM.

FIGURE 2.

PEPΔAc binds to both domains of CaM to induce a compact structure. CaM(DA) has Thr-38 (N-domain) and Thr-110 (C-domain) converted to Cys and is labeled with a sulfhydryl-specific fluorescent donor (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid) and the nonfluorescent acceptor (N-(4-dimethylamino-3,5-dinitrophenyl)maleimide). The dashed line indicates the fluorescence maximum if no FRET effect was observed.

Native Sequence of the Acidic Region Is Necessary to Modulate Ca²⁺ Binding to CaM

Table 1 shows that PEPscram has essentially no effect on K_Ca, k_off, k_on, or the stoichiometry of Ca²⁺ binding to CaM. This lack of effect was so striking that we used NMR to determine whether PEPscram binds to CaM with the same domain specificity and exchange properties as native PEP-19. We showed previously that native PEP-19 binds to apo-CaM and Ca²⁺-CaM with characteristics of slow and fast exchange, respectively, on the NMR time scale (21). Fig. 3 shows that PEPscram retains these properties. Specifically, Fig. 3a shows that PEPscram binds to apo-CaM with slow to intermediate exchange on the NMR time scale, causing severe broadening of backbone amide cross-peaks for residues in the C-domain, but it has little effect on amides in the N-domain (full spectra are supplied as supplemental material). Fig. 3b shows that PEPscram also selectively binds to the C-domain of Ca²⁺-CaM, but with characteristics of fast exchange. Thus, both PEPscram and PEP-19 bind to the C-domain of apo- or Ca²⁺-CaM, and with similar exchange characteristics, but PEPscram is incapable of modulating the Ca²⁺ binding properties of CaM.

FIGURE 3.

¹H,¹⁵N HSQC NMR spectra indicate binding of PEPscram to apo- and Ca²⁺-CaM. a shows a selected region of the overlaid ¹H,¹⁵N HSQC spectra of apo-CaM (black) and apo-CaM in complex with PEPscram (red); b shows a selected region of the overlaid ¹H,¹⁵N HSQC spectra of Ca²⁺-CaM (black) and Ca²⁺-CaM in complex with PEPscram (red). PEPscram binding causes amide resonances in Ca²⁺-CaM undergoing fast exchange on the NMR time scale. The arrows in b highlight the movements of amide resonances in Ca²⁺-CaM due to PEPscram binding.

None of the PEP-19 point mutations had significant effects on K_Ca of CaM, but Table 1 and Fig. 4a show that they have varying effects on k_off and k_on. Conversion of Glu-29 to Ala at the putative X coordination position had no effect. Mutation of Asp-31, Asp-33, or Glu-40 to Ala inhibited the ability of PEP-19 to increase k_off but to different extents. The properties of PEP(D35A) are very similar to native PEP-19, even though Asp-35 at the putative −Y coordination position is centered between residues 31, 33, and 40. This could be explained by the fact that the −Y position is highly variable in canonical EF-hand Ca²⁺-binding loops because the backbone carbonyl oxygen, not the side chain, of this residue coordinates Ca²⁺.

FIGURE 4.

Contribution of acidic residues in PEP-19 to modulating Ca²⁺ binding to the C-domain of CaM. a shows the effects of specific acidic residues in PEP-19 on the rates of Ca²⁺ dissociation (black bars) and association (white bars) at the C-domain of CaM. Dissociation of Ca²⁺ was measured in the presence of 30 μm PEP-19 or the indicated mutant PEP-19. Dissociation rates are the average of 4–5 determinations. Association rates were calculated from k_off and K_Ca by k_on = k_off/K_Ca (see Table 1). b shows the effect of increasing concentrations of PEP-19, PEP(P37G), and PEPscram on the rate of Ca²⁺ dissociation.

Fig. 4b shows that conversion of Pro-37 to Gly significantly decreased the ability of PEP-19 to modulate Ca²⁺ binding to CaM, although not to the extent seen for PEPscram. This suggests that backbone constraints imposed by the imide side chain of Pro-37 positions acidic residues in PEP-19 such that they can properly modulate Ca²⁺ binding to CaM. Therefore, mutation of Pro would be equivalent to mutating multiple acidic residues. This is consistent with the fact that PEPscram is incapable of modulating Ca²⁺ binding to CaM because it effectively has multiple acidic mutations.

Acidic Region of PEP-19 Binds Ca²⁺

The distribution of acidic residues in PEP-19 led us to determine whether the acidic sequence has intrinsic Ca²⁺ binding activity. Its similar ionic radii and metal coordination geometries to Ca²⁺ make paramagnetic Tb³⁺ a sensitive probe for identifying Ca²⁺-binding sites (26). Fig. 5a shows that Tb³⁺ broadens backbone amide chemical shifts for residues in the acidic sequence of PEP-19, especially residues 31–36, which are severely broadened at a Tb³⁺/PEP-19 ratio of 1:50. Amides for Asp-33 and Asp-35 are most affected and are broadened beyond detection at a Tb³⁺/PEP-19 ratio of 1:100. These spectral perturbations indicate that Tb³⁺ binds to the acidic region in PEP-19.

FIGURE 5.

Interactions between PEP-19 and metal ions monitored by ¹H,¹⁵N HSQC spectra. I/I₀ is the intensity ratio for backbone amide cross-peaks in the ¹H,¹⁵N HSQC spectra of 0.5 mm PEP-19 collected in the presence (I) and absence (I₀) of 10 μm Tb³⁺ (a) or 5 mm Ca²⁺ (b) in the absence of KCl. Panel c shows the effect of increasing Ca²⁺ on I/I₀ for Asp-33 and Asp-35 relative to Arg-4, which is unaffected by specific binding of Ca²⁺. * indicate the absence of backbone amides for Pro-37 and Gln-49, which could not be assigned.

Although Ca²⁺ is not paramagnetic, we reasoned that it might affect specific amide resonance intensities due to exchange broadening if Ca²⁺ binds to PEP-19. Indeed, Fig. 5b shows that addition of Ca²⁺ to PEP-19 causes exchange broadening of amide resonances in the acidic sequence relative to other regions in PEP-19. Similar to the effect of Tb³⁺, Asp-33 and Asp-35 are most affected by addition of Ca²⁺ and show maximal broadening at a Ca²⁺:PEP-19 molar ratio between 1 and 2 as shown in Fig. 5c with Arg-4 in the N-domain of CaM as a control. These spectral perturbations indicate that Ca²⁺ binds weakly to the acidic sequence of PEP-19.

Effect of Electrostatics on Ca²⁺ Binding

We reasoned that the acidic sequence of PEP-19 with intrinsic Ca²⁺ binding properties may increase the Ca²⁺ k_on if positioned near site III and/or IV of CaM by attracting or electrostatically steering Ca²⁺ to these binding sites. Because the contribution of electrostatic interactions would be decreased by monovalent cations, we predicted that decreasing the KCl concentration would increase the k_on for Ca²⁺ binding to the C-domain of CaM in the presence or absence of PEP-19. Table 2 shows the K_Ca, k_off, and k_on values for Ca²⁺ binding to the C-domain of CaM with or without 100 mm KCl and with or without 30 μm PEP-19. The Ca²⁺ binding affinity is increased about 13-fold at low ionic strength due primarily to a large increase in k_on. The effect of KCl on k_on can be explained by electrostatic shielding of acidic side chains on CaM that coordinate or attract Ca²⁺. PEP-19 increases Ca²⁺ k_on by 27- and 45-fold at 100 and 0 mm KCl, respectively. This effect of PEP-19 can be attributed, at least in part, to electrostatic steering of Ca²⁺ ions via weak Ca²⁺ binding activity of the acidic sequence in PEP-19.

TABLE 2.

Effect of ionic strength on Ca²⁺ binding to CaM

Apparent K_Ca, Hill coefficients, and k_off rates were determined as described under “Experimental Procedures.” Values are the average ± S.D. of at least three experiments. The k_on was calculated from k_on = k_off/KCa. The buffer is 20 mm MOPS, pH 7.5, with the indicated concentration of KCl. An ionic strength of 13 mm is contributed by 20 mm MOPS at this pH. PEP-19 was added to a final concentration of 30 μm.

KCl	PEP-19	KCa	Hill coefficient	koff	kon
mm		μm		s−1	μm−1 s−1
100	−	1.6 ± 0.1	1.8 ± 0.1	12.5 ± 0.1	7.8
0	−	0.12 ± 0.01	1.8 ± 0.04	7.0 ± 0.2	58
100	+	2.0 ± 0.1	1.1 ± 0.01	428 ± 40	214
0	+	0.14 ± 0.01	1.0 ± 0.05	363 ± 24	2592

PEP-19 Sensitizes HeLa Cells to ATP-induced Ca²⁺ Release

ATP-induced Ca²⁺ release in HeLa cells was selected as an initial model system to determine whether PEP-19 can impact a Ca²⁺ release pathway because this pathway involves multiple potential points of regulation by CaM, including P2Y G-protein-coupled receptors, phospholipase C, and the IP₃ receptor. PEPscram, PEP(P37G), and PEPΔAc were selected to test the biological significance of the acidic sequence because they all bind to CaM but have little or no effect on its Ca²⁺ binding properties. Expression plasmids for native and mutated PEP-19 were engineered with N-terminal Myc tags to readily determine relative expression levels in transfected cells. We anticipated that the Myc tag would not affect interactions between PEP-19 and CaM because residues 1–23 in PEP-19 are disordered when bound to CaM. As shown in Table 1, PEP-19 and mycPEP-19 have essentially identical effects on Ca²⁺ binding to CaM.

HeLa cells were transfected with a control YFP plasmid or cotransfected with YFP and PEP-19 plasmids at a 1:4 ratio. YFP-positive cells from different coverslips were selected for analysis. The Western blot in Fig. 6a shows comparable levels of expression of PEP-19 proteins in transfected cells, and it confirms that the apparent molecular mass of PEPΔAc is smaller than the other proteins due to deletion of the acidic sequence. Fig. 6b shows intracellular Ca²⁺ in response to stimulation with 0.1, 1, and 10 μm ATP. The most striking observation is that only cells expressing PEP-19 showed a robust increase in intracellular Ca²⁺ in response to 0.1 μm ATP. As summarized in Fig. 6c, control cells were unresponsive to 0.1 μm ATP, but 65% (35/54) of cells expressing PEP-19 responded with a significant increase in intracellular Ca²⁺ levels. Fig. 6d shows that peak intracellular Ca²⁺ release stimulated by 1 and 10 μm ATP was also significantly higher in cells expressing PEP-19 relative to control cells. Finally, Fig. 6e shows that PEP-19 increases the frequency of Ca²⁺ oscillations induced by 1 μm ATP relative to control cells.

In contrast to native PEP-19, Fig. 6c shows that only 5% of all cells expressing PEP(P37G), PEPscram, or PEPΔAc responded to 0.1 μm ATP with an increase in Ca²⁺. Fig. 6d shows that peak Ca²⁺ levels induced by 1 and 10 μm ATP are also significantly lower in cells expressing mutant PEP(P37G), PEPscram, or PEPΔAc relative to PEP-19. Moreover, Fig. 6d shows the mutant PEP-19 proteins do not mimic the effect of native PEP-19 on Ca²⁺ oscillation frequency at 1 mm ATP. These data demonstrate that simply binding CaM is not sufficient and that the native acidic sequence in PEP-19 is required to sensitize HeLa cells to ATP-dependent Ca²⁺ release.

DISCUSSION

Cell signaling pathways must be regulated at multiple levels to control the amplitude and temporal characteristics of cellular responses and to prevent chaotic signaling that can lead to cell damage or death. Calmodulin is primarily regulated by intracellular Ca²⁺, which is in turn controlled by cell-specific arrays of Ca²⁺ channels, pores, and pumps (27). A poorly understood regulatory mechanism involves the actions of dedicated regulators of CaM signaling, which have no known intrinsic activity other than binding to CaM. For example, the small neuronal phosphoprotein called ARPP-21, or regulator of calmodulin signaling, binds to Ca²⁺-CaM to competitively inhibit activation of calcineurin and block suppression of L-type Ca²⁺ currents (28). PEP-19 is also a small protein with the potential to broadly affect CaM signaling by binding to apo- or Ca²⁺-CaM via its IQ motif.

An obvious potential mechanism for PEP-19 is to competitively inhibit activation of CaM targets as proposed in the camstatin model (12). A caveat to this is that enzymes such as CaM kinase II bind CaM with 10,000-fold greater affinity than does PEP-19. Nevertheless, CaM binds to many proteins with low affinity, and PEP-19 would be particularly effective as an antagonist of proteins that bind preferentially to the apo- or Ca²⁺-bound C-domain of CaM. Another mechanism, the calpacitin model (14), proposes that higher affinity binding of PEP-19 to apo-CaM relative to Ca²⁺-CaM retards its release during a Ca²⁺ pulse thereby affecting the temporal profile of available CaM and decreasing the overall rate of association of CaM with Ca²⁺-dependent target proteins, especially at low Ca²⁺ levels. This model stems from early studies showing that the homologous protein, neuromodulin, binds preferentially to apo-CaM (29). However, this selectivity is only observed at low salt, and neuromodulin binds with equal affinity to apo- and Ca²⁺-CaM in buffers containing 150 mm KCl (30). PEP-19 also has little selectivity for apo- versus Ca²⁺-CaM at physiologically relevant concentrations of salt (12, 16).

These caveats to proposed mechanisms for PEP-19 led us to explore alternatives. We first showed that PEP-19 increased k_on and k_off rates for Ca²⁺ binding to the C-domain of CaM by 30–40-fold without greatly affecting K_Ca (15). Importantly, an acidic sequence located adjacent to the IQ motif is required for PEP-19 to modulate Ca²⁺ binding to CaM (16). Thus, PEP-19 has the potential to modulate the rate-limiting kinetics of Ca²⁺ binding to CaM and to provide a regulatory mechanism that is analogous to regulators of G-protein signaling, or RGS proteins, that modulates nucleotide hydrolysis (31).

The first goal of this study was to investigate the molecular mechanism of action of PEP-19. Our results show the following: 1) the native sequence of the acidic region as well as backbone constraints imposed by Pro-37 are required for PEP-19 to modulate Ca²⁺ binding to CaM; 2) the acidic sequence has weak Ca²⁺ binding properties. Interestingly, mutations that compromise the ability of PEP-19 to modulate Ca²⁺ binding to CaM have proportional effects on both k_on and k_off (see Table 1), which suggests that a similar mechanism is responsible, at least in part, for modulating both parameters. A role for acidic residues in tuning the Ca²⁺ k_on but not k_off for binding to Ca²⁺ EF-hand proteins was demonstrated by Martin et al. (32), who showed that neutralizing three acidic surface residues near EF loop I in calbindin D9k decreased the k_on up to 50-fold. By analogy, the acidic sequence of PEP-19 may mimic an increase in negative surface charge near site III and/or IV of CaM, thereby increasing the Ca²⁺ k_on CaM by stabilizing a Ca²⁺-CaM initiation complex or by electrostatically steering Ca²⁺ to sites III and/or IV. PEP-19 may increase the Ca²⁺ k_off of CaM by providing a low affinity transition Ca²⁺-binding site that shuttles Ca²⁺ to the solvent rather than allowing it to rebind to the EF-hands of CaM. The inability of PEP(P37G) and PEPscram to modulate Ca²⁺ k_on and k_off may be due to repositioning the acidic residues relative to the EF-hand Ca²⁺-binding loops in CaM and/or compromising Ca²⁺ binding to PEP-19.

The second goal of this study was to determine whether PEP-19 modulates CaM-dependent signaling pathways that affect intercellular Ca²⁺ homeostasis. We selected purinergic ATP-induced Ca²⁺ release as a model system because this pathway involves multiple potential points of regulation by CaM. The data in Fig. 6 show that PEP-19 sensitizes HeLa cells to ATP-dependent Ca²⁺ release and also alters the frequency of Ca²⁺ oscillations. Importantly, these biological effects require an intact acidic sequence, not simply binding of PEP-19 to CaM. Additional studies will be necessary to identify the level at which PEP-19 impacts Ca²⁺ release, but these effects reinforce the idea that both PEP-19 and Ng play roles in intercellular Ca²⁺ homeostasis (33). Such a role would be consistent with expression of PEP-19 in neuroendocrine and neuronal cells such as Purkinje cells (34) that have highly active Ca²⁺ signaling dynamics with robust and prolonged trains of action potentials (35). Ng knock-out mice show multiple effects on Ca²⁺ dynamics, including increased base-line Ca²⁺ levels and blunted Ca²⁺ transients induced by synaptic activity or glutamate receptor agonists (36). We anticipate PEP-19 and Ng will influence distinct sets of Ca²⁺ mobilization proteins and/or have different effects on the same proteins because PEP-19 increases both k_on and k_off of Ca²⁺ binding to the C-domain (15), whereas Ng increases only Ca²⁺ k_off leading to decreased Ca²⁺ binding affinity (37). Different cellular effects of PEP-19 and Ng are also suggested by different patterns of expression and because PEP-19 has anti-apoptotic effects (9, 10), whereas RC3 is reported to have pro-apoptotic activity (38, 39).

Calmodulin regulates numerous proteins involved in Ca²⁺ mobilization that could be tuned by PEP-19. With respect to ATP-dependent Ca²⁺ release, CaM directly and indirectly impacts phospholipase C activity (40), and it also modulates the activity of the IP₃ receptor (41) and store-operated Ca²⁺ entry channels (42) subsequent to IP₃ generation. Other CaM-dependent channels and extrusion proteins include the ryanodine receptor (43), plasma membrane Ca²⁺ pumps (44), and the Na⁺/Ca²⁺ exchanger (45). Interestingly, the modes of interaction between CaM and several key Ca²⁺ mobilization proteins may make them particularly susceptible to PEP-19 because it binds selectively to the C-domain of CaM. For example, voltage-operated Ca²⁺ channels (46) and the IP₃ receptor (47) rely on selective, sequential, or stepwise interactions with the C-domain of CaM in its apo- or Ca²⁺-bound forms.

In summary, this study reveals new mechanisms of action for PEP-19 and demonstrates novel effects on ATP-dependent Ca²⁺ release that do not depend solely on binding PEP-19 to CaM, but it also requires its ability to modulate Ca²⁺ binding to CaM. Tuning the activities of Ca²⁺ mobilization pathways would place PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.