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J. Biol. Chem., Vol. 278, Issue 49, 49589-49599, December 5, 2003
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From the
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3200 and the ¶Universität Hamburg, Zentrum für Molekulare Neurobiologie, Institut für Neurale Signalverarbeitung, Falkenried 94, 20251 Hamburg, Germany
Received for publication, August 14, 2003 , and in revised form, September 25, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-helix and that frequenins bind to one side of this -helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In the structures of both human NCS-1 and yeast Frq1, the four EF-hands come in two pairs positioned in a tandem linear array on one side of the molecule. This arrangement is quite different from that found in calmodulin and troponin C, where each pair of EF-hands is situated at either end of a dumbbell-shaped molecule (5). The overall topology of two pairs of EF-hands in a tandem linear array is also observed in the structures of other members of the N-myristoylated, small Ca2+-binding proteins, namely neurocalcin (6), recoverin (7, 8), and GCAP (9). Despite this superficial similarity, both yeast Frq1 (3) and human NCS-1 (2) possess a wide hydrophobic crevice at their surface situated between the two EF-hand-containing lobes, as well as solvent-exposed N and C termini. These latter features are lacking in recoverin, neurocalcin, and GCAP.
The function of frequenin has attracted much attention ever since it was implicated in neurotransmitter release at neuromuscular junctions. In fact, the name of the molecule derives from the fact that mutants of D. melanogaster that overexpress this protein display a marked facilitation of neurotransmitter release in third instar larvae, which depends dramatically on the frequency of stimulation (10). On this basis, it was suggested that frequenin might serve as a calcium sensor to modulate synaptic activity and secretion. Likewise, in a tissue culture cell model of synaptic transmission, overexpression of rat NCS-1 in live PC12 cells potentiated release of growth hormone-containing secretory vesicles in response to agonists such as ATP (11, 12). Indirect immunofluorescence indicates that NCS-1 is localized at the trans-Golgi network of animal cells, consistent with a role for frequenin in vesicle trafficking and exocytosis (2). Several lines of evidence suggest, however, that frequenin-mediated modulation of exocytosis may be exerted at the level of signal transduction, rather than through direct effects on the machinery for exocytosis. For instance, Ca2+-induced exocytosis in permeabilized PC12 cells is not potentiated by overexpression of NCS-1 (13). Similarly, NCS-1 is apparently not involved in modulation of depolarization-evoked exocytosis in PC12 cells (13). By contrast, like the enhancement of agonist-evoked exocytosis in PC12 cells by NCS-1 overexpression, agonist-induced phosphoinositide turnover is also potentiated when NCS-1 is overexpressed (13, 14). These data suggest that NCS-1 might modulate some aspect of phosphoinositide metabolism in animal cells, thereby promoting, for example, agonist-activated phospholipase C-dependent signaling. In agreement with this hypothesis is the observation that NCS-1 and phosphatidylinositol (PtdIns) 4-kinase-
co-localize in animal cells, as judged by immunocytochemical approaches (2, 14).
Compelling evidence for a role of frequenin in phosphoinositide metabolism was first found by a combination of genetic and biochemical studies in the yeast S. cerevisiae (1). It was shown that the yeast ortholog of frequenin, Frq1, functions as a Ca2+-sensing subunit of the yeast PtdIns 4-kinase isoform, Pik1. Pik1 is a key enzyme in the control of vesicular trafficking in the late secretory pathway (15–17). Absence of Pik1 in S. cerevisiae cells (a pik1
null mutation) is lethal (18), and pik1-ts mutants exhibit severe protein trafficking defects and accumulate morphologically aberrant Golgi membranes at the restrictive temperature (15–17). Likewise, frq1 mutants are inviable, but overexpression of PIK1 suppresses the lethality of frq1 cells, indicating that Pik1 is the sole essential target for Frq1 in yeast cells (1). Yeast Frq1 co-purifies stoichiometrically with Pik1 and is required for optimal activity of the enzyme and may also assist in membrane recruitment of Pik1 (1, 3, 19). Frq1 and Pik1 form a 1:1 complex, and the binding site for Frq1 has been mapped to residues 125–169 within the N terminus of Pik1 (20). The binding of Frq1 to Pik1 appears to be mediated primarily by hydrophobic interactions in a Ca2+-independent manner, as judged by a variety of different biochemical studies (1, 20).
Given that the primary and tertiary structures of frequenins have been highly conserved from yeast to man, we sought to investigate in this study how closely the function of human NCS-1 resembles that of yeast Frq1. In a first approach, we examined whether human NCS-1 is able to functionally substitute for the absence of Frq1 in yeast and, if so, whether Ca2+ binding is required for the ability of human NCS-1 to rescue the inviability of Frq1-deficient yeast cells. Second, we used biochemical approaches, including the two-hybrid method in vivo and pull-down assays and monitoring of intrinsic tryptophan fluorescence in vitro, to assess whether human NCS-1 can interact with yeast Pik1. If so, we sought to determine whether NCS-1 associates at the same site on Pik1 as Frq1, then to delineate the sequence determinants responsible for this binding using alanine- and lysine-scanning mutagenesis, and finally to measure the strength and stoichiometry of this binding.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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NCS-1 and PIK1 Cloning, Expression, and Purification—Expression and purification of NCS-1 have been described previously (2). DNA encoding PIK1 was amplified by PCR using yeast genomic DNA as the template with primer sequences, derived from the data base entry for PIK1 (GenBankTM/EBI accession no. NC_001146
[GenBank]
), designed to permit subcloning into pET22b (Novagen) that was linearized with NdeI and SacI, generating the bacterial expression vector pET-PIK1. pET-PIK1 was introduced into E. coli strain BL21(DE3)RIL (Stratagene) by DNA-mediated transformation. The resulting cells were grown in Luria-Bertani medium containing ampicillin (100 µg/ml) and chloramphenicol (50 µg/ml) at 37 °C until the culture reached an A600 nm of 0.7. Next, Pik1-His6 expression was induced by addition of isopropyl-1-thio--D-galactopyranoside to a final concentration of 0.5 mM. Induced cells were harvested by centrifugation, resuspended in 20 ml of lysis buffer (20 mM Tris/HCl, pH 7.9, 300 mM NaCl) per 500 ml of bacterial culture and subsequently treated with lysozyme (final concentration 100 µg/ml) for 10 min on ice. Dithiothreitol (DTT) and N-laurylsarcosine were added to final concentrations of 5 mM and 1.5% (v/v), respectively. After sonication (1 min, level 4, 50% duty cycle), the lysate was cleared of cellular debris by centrifugation (10,000 x g, 10 min, 4 °C). The resulting supernatant solution was adjusted to a final concentration of 3% Triton X-100, filtered (0.45-µm pore size) and loaded onto a 1-ml Ni2+-saturated nitrilotriacetic acid (NTA)-agarose column (Qiagen). After washing with buffer A (20 mM Tris/HCl, pH 7.9, 300 mM NaCl, 20 mM imidazole), bound protein was eluted with buffer B (20 mM Tris/HCl, pH 7.9, 300 mM NaCl, 250 mM imidazole). Pik1-His6-containing fractions were dialyzed against 20 mM Tris/HCl, pH 7.9 and concentrated with the help of centrifugal filter devices (Millipore) until a final concentration of 
5 mg/ml had been reached.
In Vitro Pull-down Assays—NCS-1 (0.7 µg), expressed in and purified from bacteria (2), was incubated at 4 °C either alone or with 7 µg of the different Pik1-His6 derivatives in a final volume of 0.5 ml of YLB (50 mM Tris/HCl, pH 7.2, 100 mM NaCl, 1 µM CaCl2, 1 mM DTT) on a roller drum for 1 h, and then 20 µl of a 50% slurry of Ni2+-saturated NTA-agarose beads (Qiagen) that had been preblocked in YLB containing 1% (w/v) bovine serum albumin were added. After an additional 1 h of incubation at 4 °C, the beads were washed three times (1 ml each) in wash buffer (20 mM Tris/HCl, pH 7.9, 300 mM NaCl, 10 mM -mercaptoethanol, 1 µM CaCl2, 50 mM imidazole). Subsequently, bound proteins were eluted from the beads in 30 µl of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) sample buffer (28), resolved by electrophoresis in 15% SDS-PAGE gels, and analyzed by immunoblotting using appropriate antisera or staining with Coomassie Brilliant Blue.
Yeast Strains, Growth Conditions, and Media—Routine growth and manipulation of S. cerevisiae strains were performed as described previously (29). Conventional methods for DNA-mediated transformation of yeast cells were used (30). Strain YKBH1 has been described elsewhere (1). All yeast strains used in this study are listed in Table I.
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Preparation of Yeast Cell Extracts and Immunoblot Analysis—Yeast cells were grown at 30 °C in standard rich (YP) medium, containing 2% galactose as carbon source (29). Preparation and preclearing of cellular lysates were carried out essentially as described (20). Protein concentration in each clarified extract was determined using a commercial kit (Bio-Rad) based on the dye-binding method of Bradford (31). Equal amounts of protein were resolved in 12.5% SDS-PAGE gels, transferred onto nitrocellulose membrane, and wild-type and mutant versions of NCS-1 were detected by immunoblotting using affinity-purified polyclonal NCS-1 antiserum (dilution 1:500), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG conjugate (Zymed) and a commercial chemiluminescence kit (Western Lightning, PerkinElmer Life Sciences).
Yeast Two-hybrid Assay—Two-hybrid experiments were performed using yeast strain CG-1945 (Clontech), harboring HIS3 and lacZ (-galactosidase) reporter genes. pGAD424-based (Gal4 transcriptional-activation domain fusion vector, Clontech) derivatives of PIK1 and NCS-1, as well as pAS2–1 (Gal4 DNA binding domain fusion vector, Clontech) constructs of KChIP2, calmodulin, NCS-1, and PIK1 were used. Transformations and subsequent steps were performed according to the instructions from the manufacturer (Matchmaker two-hybrid system, Clontech).
Intrinsic Tryptophan Fluorescence—Fluorescence spectra were recorded at room temperature using a PerkinElmer 2550B spectrofluorimeter at an excitation wavelength of 280 nm. Intrinsic fluorescence of NCS-1 (75 nM) was monitored over an emission wavelength range of 310 to 400 nm. Fluorometric titrations were done in 50 mM HEPES, pH 7.5, 0.1 M KCl, 1 mM CaCl2, and 1 mM DTT, using a 5 x 10-mm black cuvette. Fluorescence spectra were recorded before and after the addition of 2.5–40 µl of Pik1(120–192) (final concentration 20–640 nM). In parallel, tryptophan emission spectra of comparable fluorescence intensity were collected at 340–360 nm to correct for dilution and unspecific effects of Pik1(120–192) absorbance. The Pik1 peptide itself does not contain any tryptophan residues and, therefore, should not contribute any fluorescence to affect these measurements. Binding data were calculated from the observed changes in fluorescence intensity accompanying the addition of Pik1(120–192), using methods described previously (32). Data analysis and curve fitting were done with the help of KaleidaGraph software (Synergy Software). For KD measurements, titrations were performed at protein concentrations below the KD value. Values were determined assuming that all proteins present were in their native and functional state and not corrected for the possible presence of inactive species.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cells Is Rescued by Human NCS-1—Frequenins seem to be conserved at the level of both primary and tertiary structure from yeast to humans. Frq1, the apparent S. cerevisiae ortholog of NCS-1, binds to and is essential for the function of Pik1, a PtdIns 4-kinase, and this role is the only indispensable cellular function of Frq1 (1). Whether NCS-1 directly interacts with a PtdIns 4-kinase in vertebrate cells, and whether this association is physiologically relevant, has not been demonstrated unequivocally, although considerable circumstantial evidence has been obtained in support of this conclusion (14, 33, 34). As one approach to address this question, we examined whether NCS-1 is able to functionally substitute for Frq1 in S. cerevisiae cells. To this end, a heterozygous diploid strain, YKBH1 (frq1::HIS3/FRQ1) (Table I), was transformed with a LEU2-marked 2-µm-based (high copy) vector expressing human NCS-1 cDNA under control of the inducible GAL1 promoter. The resulting transformants were sporulated, and ascospores were dissected on YP plates containing galactose to induce NCS-1 expression and then incubated at a temperature (30 °C) normally optimal for yeast cell growth. The majority of dissected tetrads yielded four viable spores (Fig. 1A, left panel), and His+ Leu+ spores were obtained at the expected frequency. Thus, NCS-1 is able to functionally replace Frq1 in yeast cells lacking any endogenous Frq1.
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To investigate the roles of N-myristoylation and Ca2+ binding for NCS-1 function in vivo, the ability of mutant versions of NCS-1 to rescue the inviability of frq1 cells was tested in the same fashion. Both a non-myristoylated mutant, NCS-1(G2A) and a mutant in which the Ca2+ binding affinity of the three functional EF-hands had been crippled, NCS-1(E81V T117A T165A) (2), rescued the lethality of the frq1 mutation, indicating that neither N-myristoylation nor Ca2+ binding are essential for NCS-1 function (Fig. 1A, middle panels). These results were not unexpected because it had been demonstrated previously that N-myristoylation of Frq1 is dispensable for its function and that Frq1 associates with Pik1 tightly even in the absence of Ca2+ (1). As judged by immunoblotting with polyclonal anti-NCS-1 antibodies, absence of N-myristoylation increased the steady state level of NCS-1 in both the initial diploid strain (YKBH1) (Fig. 1B, upper panel) and in the derived frq1 cells carrying the same plasmid (Fig. 1C).2 Conversely, the Ca2+ binding-defective mutant was reproducibly expressed at a level distinctly lower than normal NCS-1 in both YKBH1 (Fig. 1B, upper panel) and in the derived frq1 cells carrying the same plasmid (Fig. 1C), suggesting that Ca2+ binding helps stabilize the protein. Remarkably, even a quadruple mutant, NCS-1(G2A E81V T117A T165A), also supported the growth of cells lacking Frq1 (Fig. 1A). This result was somewhat surprising because it had been shown before that Ca2+ binding induces major structural changes in Frq1 that contribute independently of N-myristoylation to its ability to associate with membranes (3). However, the NCS-1(E81V T117A T165A) protein does have detectable residual Ca2+ binding activity (2). Moreover, just as observed with otherwise wild-type NCS-1, lack of N-myristoylation significantly increased the apparent steady state level of the NCS-1(E81V T117A T165A) protein in both YKBH1 (Fig. 1B, upper panel) and in the derived frq1 cells carrying the same plasmid (Fig. 1C). These properties could account for the observed ability of the quadruple mutant to suppress the inviability of frq1 cells.
As one means to distinguish whether all of the NCS-1 variants retained full function, we examined their ability to rescue the inviability of frq1 cells under the somewhat stressful condition of elevated temperatures (35, 36, and 37 °C) and when the level of expression was reduced (on glucose medium, which represses expression from the GAL1 promoter). The frq1 strains carrying each of the plasmids (His+ Leu+ cells), derived from the tetrad dissections of YKBH1 carrying the corresponding plasmid, were streaked out to single colonies on either YPGal and YPD plates and incubated at various temperatures. An isogenic yeast strain, YKBH7 (frq1::HIS3) (Table I), expressing FRQ1 from an URA3-marked high copy plasmid, was used as a positive control. When grown on YPGal plates, there were only modest differences in growth between wild-type NCS-1 and its mutant versions at all temperatures tested up to 36 °C (Fig. 1D and Table II). However, when the cells were grown on YPD, only NCS-1 (YTS47) and NCS-1(G2A) (YTS48), but not NCS-1(E81V T117A T165A) (YTS51) or NCS-1(G2A E81V T117A T165A) (YTS53), were able to support growth at 30 °C (Fig. 1D and Table II). At higher temperatures, even NCS-1 and NCS-1(G2A) were unable to support vigorous growth, especially compared with the robust growth displayed by frq1 cells expressing authentic Frq1 (Fig. 1D and Table II). Nevertheless, NCS-1 and the quadruple mutant NCS-1(G2A E81V T117A T165A) displayed very similar expression levels (Fig. 1, B and C), yet distinctly different phenotypes (Fig. 1D and Table II), arguing that the differences in their observed behavior cannot be attributed to differences in their level of expression. Thus, by these criteria, we conclude, first, that the loss of Ca2+ binding clearly compromises the function and/or structural integrity of NCS-1 at higher temperatures, which can be largely compensated for by elevated expression. Second, although NCS-1 is clearly able to substitute for Frq1, even wild-type NCS-1 is not completely equivalent to native yeast Frq1.
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-NCS-1 antiserum and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin. As observed in previous studies with yeast Frq1 (1, 20), there was little or no nonspecific binding of human NCS-1 to empty beads (Fig. 2B, lane 4). In contrast, when the beads were first decorated with Pik1(10–192)-His6, NCS-1 bound with high efficiency (Fig. 2B, lane 1). Moreover, this binding could be effectively competed by addition of a 10-fold molar excess of yeast Frq1 (Fig. 2B, lane 2), but not by an equivalent amount of KChIP2 (Fig. 2B, lane 3). These data confirm that NCS-1 is able to associate specifically and directly with the N terminus of Pik1 in the absence of any other yeast protein and demonstrate that the site bound must be congruent, or overlap substantially, with that normally occupied by Frq1. Moreover, as also observed for Frq1-Pik1 interaction, association of NCS-1 with Pik1 was not dependent on Ca2+ because equivalent binding of NCS-1 to Pik1(10–192)-His6-coated beads was observed in either the presence of 1 mM Ca2+ or when a 2-fold molar excess of EGTA was present (Fig. 2C). Mapping the NCS-1-binding Region in Pik1—A 44-residue segment of Pik1 (positions 125–169) is both necessary and sufficient for its efficient interaction with Frq1 (20). To delineate the region within Pik1(10–192) (designated "Fragment a" or Fa) responsible for its association with NCS-1, we first generated several overlapping deletion derivatives (Fig. 3A). One fragment (Fb) represented the most proximal portion of the Pik1 N terminus (residues 10–107) and contained a sequence element referred to as the "lipid kinase unique domain," which is conserved among PtdIns 3- and PtdIns 4-kinases (18, 36–39). The second fragment (Fc) corresponded to the central region (residues 53–144) of Pik1(10–192), and the third fragment (Fd) contained the C-terminal segment (residues 100–192) of Pik1(10–192) (Fig. 3A). Interaction of these fragments with NCS-1 was examined, first, using the yeast two-hybrid reporter assay. For this purpose, the coding sequence for each fragment was introduced into vector pAS2–1, creating the corresponding chimeras with the Gal4BD. The resulting baits were tested for their ability to interact with Gal4AD-NCS-1 (in pGAD424-NCS-1). Only full-length Pik1(10–192) (Fa) and its C-terminal end (Fd), corresponding to residues 100–192, were able to associate with NCS-1, as judged by growth of the reporter strain CG-1945 (Fig. 3B).
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As an independent means to verify these conclusions, four of the fragments (Fa, Fb, Fe, and Ff) were tagged with C-terminal His6 tracts and the corresponding derivatives were expressed in and purified from E. coli. Ni2+-saturated NTA beads coated with these purified fragments were then used for in vitro pull-down assays. After incubation with purified NCS-1 (or with purified Myc epitope-tagged Frq1, as a positive control) and thorough washing, the bead-bound proteins were resolved by SDS-PAGE and analyzed by immunoblotting with appropriate antibodies to detect NCS-1 and Frq1 and by staining with Coomassie Brilliant Blue to determine the quantity of each His6-tagged fragment that had been immobilized on the beads. As observed before, little or no NCS-1 bound to empty beads (Fig. 3C, lane 5). Likewise, Fb, which did not interact with NCS-1 in the two-hybrid assay, was also unable to bind NCS-1 in vitro (Fig. 3C, lane 2). Similarly, Fc was also unable to bind NCS-1 in the in vitro pull-down assay (data not shown). As expected, NCS-1 bound avidly to Fa. Most importantly, and in agreement with the results obtained by the two-hybrid method, both Fe and Ff were able to bind NCS-1 with an affinity similar to NCS-1 binding by Fa (Fig. 3C, lanes 3 and 4). Essentially identical results were obtained for the binding of Frq1 to the same set of fragments (Fig. 3D).
Mapping the NCS-1-binding Residues in Pik1 by in Vitro Mutagenesis—Binding of Frq1 to Pik1 seems to depend largely on hydrophobic interactions (20), and both NCS-1 (2) and Frq1 (3) have a wide hydrophobic crevice at their surface. These considerations suggested that hydrophobic side chains within the 145–172 segment of Pik1 might contribute primary contacts that mediate the docking of NCS-1. As one means to characterize the chemical nature of the interaction of NCS-1 with Pik1(10–192), we performed Ala and/or Lys scanning mutagenesis at 39 positions within the NCS-1-binding region of Pik1(10–192) (Table III and Fig. 4A). To examine the ability of these mutants to bind purified NCS-1, each was tagged at its C terminus with His6 and expressed and purified from E. coli and used for the in vitro pull-down assay, as described above. None of the Ala substitution mutations tested was impaired in its ability to interact with NCS-1 (Fig. 4B and Table III). In contrast, replacement of some of the same residues with Lys either reduced somewhat (S164K and, perhaps, I170K) or almost totally eliminated (L160K and A171K) NCS-1 binding (Fig. 4B and Table III). These effects were most dramatically illustrated for double mutants. Pik1(10–192; L160A S164A) bound NCS-1 with an affinity indistinguishable from normal Pik1(10–192), whereas Pik1(10–192; L160K S164K) showed no detectable binding of NCS-1 whatsoever (Fig. 4C). These observations confirm in striking fashion that the hydrophobic character of residues in this region of Pik1 are crucial for NCS-1 binding. In addition, given the spacing of the residues that appear to be most critical for NCS-1 binding, as judged by the Lys scanning mutagenesis, the results would be most easily interpreted if these residues projected from the same face of an -helix, suggesting that part of the NCS-1 binding site in Pik1 (at least that situated between residues 160 and 171) may adopt an -helical conformation and, therefore, that NCS-1 binds to one side of this -helix. Recent NMR studies on complexes of Frq1 with Pik1-derived peptides indicate that a similar 13-residue stretch of Pik1 (residues 157–169) comprises part of the Frq1-binding site (20) and adopts an -helical conformation.3
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Monitoring of Pik1-NCS-1 Interaction by Fluorescence Spectroscopy—To detect possible structural changes induced in NCS-1 upon its binding to Pik1, we monitored the intrinsic tryptophan fluorescence of NCS-1. The NCS-1 polypeptide contains just two Trp residues: Trp30, adjacent to EF1, and Trp103, neighboring EF3. Both of these Trp residues are invariant features of frequenins from yeasts (e.g. S. cerevisiae, Schizosaccharomyces pombe, Candida albicans), invertebrates (e.g. C. elegans, D. melanogaster), non-mammalian vertebrates (e.g. Xenopus laevis), and mammals (e.g. mice, rats, humans). For these studies, we used Pik1(120–192) because it contains the NCS-1 binding site, but lacks any Trp residue, and, as a consequence, should not contribute to the fluorescence emitted at the wavelengths examined. To monitor structural changes in NCS-1 induced by its interaction with Pik (120–192), corresponding changes in the intensity of the fluorescence emission were monitored. Upon excitation at 280 nm, the fluorescence emission spectrum of free Ca2+-bound NCS-1 exhibited a maximum at 340 nm. Addition of a 2-fold molar excess of Pik1(10–192)-His6 increased the emission at 340 nm by 20% and caused the emission maximum to shift by 5 nm to the blue (Fig. 5A). The change in the intensity of the fluorescence at 340 nm as a function of increasing concentration of Pik1(120–192) was used to measure the apparent affinity of NCS-1 for the peptide. As described in detail under "Experimental Procedures," a saturation binding curve can be calculated from the observed changes in fluorescence intensity (Fig. 5B). From these binding data (three independent trials), one can calculate both the Hill coefficient, which was 1.01, and a dissociation constant (KD = 0.15 ± 0.01 µM). These results further corroborate that NCS-1 binds to Pik1(120–192) with high affinity and non-cooperatively, most likely indicative of a 1:1 stoichiometry for the complex.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Increases in mammalian NCS-1/frequenin in organisms with a nervous system enhance neurotransmitter release at neuromuscular junctions (10, 41) and at synapses of the central nervous system (42). There are data showing that frequenin action promotes both agonist-induced potentiation of N-type Ca2+ channels (43) and activity-dependent facilitation of currents through P/Q-type Ca2+ channels (42), suggesting, first, that frequenin contributes to synaptic facilitation at a variety of different synapses and, second, that enhanced Ca2+ entry might explain the increase in synaptic vesicle release when NCS-1 is overexpressed. The apparent modulation of oocytes-expressed A-type K+ channel activity by NCS-1 (40) might also represent an indirect effect of changes in cellular Ca2+. On the other hand, it has been reported that overexpression in bovine chromaffin cells of a purported dominant-negative mutant, NCS-1(E120Q), increased (rather than decreased) non-L-type Ca2+ channel currents (44). In addition to the evidence for some regulatory role of NCS-1 that affects the function of ion channels, NCS-1 has also been implicated in Ca2+-signaling pathways that result in receptor desensitization by G-protein-coupled receptor kinases (45–47). Correspondingly, in PC12 cells, NCS-1 overexpression increases phosphoinositide turnover in response to agonists, such as UTP and bradykinin, that are known to activate receptors that stimulate phospholipase C, presumably resulting in inositol trisphosphate-dependent Ca2+ release, thereby leading to increased Ca2+-dependent events and enhancement of exocytosis (13). Taken together, these data suggest that the effects of NCS-1/frequenin on ion channel activity, Ca2+ signaling, exocytosis/neurotransmitter release, and the efficiency of synaptic function, are all indirect. All of these effects could be explained if NCS-1 controlled the supply of phosphoinositides (48), a premise based largely upon what is known about the molecular function of Frq1, the NCS-1 ortholog in budding yeast (S. cerevisiae).
It has been shown that Frq1 is bound tightly to, and serves as a non-catalytic subunit of, the essential PtdIns 4-kinase isoform, Pik1 (1). At restrictive temperature, pik1ts mutants display defects in the delivery of secretory proteins from the Golgi apparatus to the plasma membrane (15–17), suggesting a role for Pik1-derived phosphoinositides in a late stage of the secretory pathway. In agreement with this finding is the demonstration that pik1 mutants accumulate abnormal Golgi structures, termed "Berkeley bodies," at the restrictive temperature. Frq1, which is itself essential for yeast cell viability, co-purifies stoichiometrically with Pik1 from yeast cell extracts and is required for optimal activity of the enzyme (1). Moreover, Frq1 is able to interact with biological membranes via both its N-myristoyl group and via residues exposed upon the conformation changes induced by Ca2+ binding (3). Thus, Frq1 might assist with recruitment of Pik1 to membranes, because Pik1 itself lacks any obvious membrane targeting motifs. Pik1 is the yeast ortholog of mammalian PtdIns 4-kinase (PtdIns4K), an enzyme also found to associate primarily with the Golgi apparatus (36). Hence, it is conceivable that NCS-1 regulates PtdIns4K in mammalian cells in a manner analogous to the modulation of Pik1 activity by Frq1 in yeast. Indeed, over time, there have been reports that NCS-1 and PtdIns4K can be co-immunoprecipitated from lysates of several tissue culture cell lines, including bovine chromaffin cells, Madin-Darby kidney cells, and COS-7 cells (14, 49, 50), suggesting that NCS-1 and PtdIns4K might interact directly. Consistent with this notion, overlapping immunocytochemical localization also has been reported for NCS-1, the small GTPase, ARF1, and PtdIns4K in COS-7 cells (2) and for NCS-1 and PtdIns4K in newborn cultured dorsal root ganglia neurons (51).
All frequenins, including NCS-1, are myristoylated at their N termini (12), allowing them to transiently interact with biological membranes. Irreversible covalent attachment of myristate requires an N-terminal Gly residue (52). When the non-myristoylatable NCS-1(G2A) mutant was present as the sole source for frequenin in frq1 cells, we could detect no phenotypic differences from frq1 cells expressing wild-type NCS-1. Thus, N-myristoylation of NCS-1 is not essential for its function, at least in yeast cells, in good agreement with the previous finding that a Frq1(G2A) mutant also restores viability to frq1 cells (1).
Frq1 and NCS-1 are both able to coordinate three Ca2+ ions per molecule with high affinity (2, 3). In prior work, based on both biochemical and genetic approaches, optimal membrane association of Frq1 was found to depend on both Ca2+-binding and the N-terminal myristoyl group (3), but no mutations in the EF-hand domains of Frq1 were generated to investigate the role of Ca2+ binding in its cellular function. In this regard, we found that NCS-1(E81V T117A T165A), in which the Ca2+ binding affinity of all three functional EF-hand domains has been greatly impaired (2), was still able to support growth of frq1 cells. This finding was somewhat unexpected because it was shown by a variety of biophysical techniques that Ca2+ binding induces major structural changes in Frq1 (3) and, presumably, also in NCS-1. However, NCS-1(E81V T117A T165A) does display residual Ca2+ binding activity (2), which might explain how it was still able to function when it was expressed at a high level from the GAL1 promoter. However, at low levels of expression, NCS-1(E81V T117A T165A) was not capable of restoring viability in frq1 cells under conditions where normal NCS-1 did. By this criterion at least, the lack of efficient Ca2+ binding does compromise the function, folding, and/or stability of NCS-1 and/or in its ability to assist with the targeting of Pik1 to membranes.
Some members of the family of small, N-myristoylated, Ca2+-binding proteins undergo a dramatic change in conformation upon Ca2+ binding that extrudes the N-myristoyl group from a pocket buried in the protein and places it in a solvent-exposed condition, a structural rearrangement dubbed the "Ca2+-myristoyl switch" (8, 53–56). Several lines of evidence indicate that frequenins do not possess this Ca2+-myristoyl switch mechanism. First, as judged by a wide range of biophysical experiments, the myristoyl group in Frq1 and NCS-1 appears to be constantly solvent-exposed (2, 3). Second, Frq1 and NCS-1 have a high affinity for Ca2+, with a KD not far above the value (100 nM) for the basal level of cellular Ca2+ concentration, suggesting that a large fraction of the population of frequenin molecules might be in the Ca2+-bound state, even in resting cells. Finally, as shown here for NCS-1 and earlier for Frq1 (1), the fact that N-myristoylation is dispensable for cellular function argues against regulation of Frq1 membrane association via a Ca2+-induced myristoyl switch.
Prior work demonstrated that Frq1 interacts with the N terminus of Pik1 (1) and does so at a specific region that includes residues 125–169 (20), which lies outside of the conserved lipid kinase unique domain motif, a sequence element that still has no defined molecular function. The Frq1-binding site in Pik1 appears to consist of two subsites defined by Phe125–Gln136 and Ala157–Ser169 (20). The distal residues bind Frq1 independently of the proximal residues, whereas binding to residues 125–136 appears to require that contact be made with residues 157–169. The minimal NCS-1-binding element in Pik1 defined by the in vitro pull-down experiments and the in vivo yeast two-hybrid reporter assays is located between residues 145 and 172, which is almost congruent with the distal element responsible for Frq1 binding. Presence of residues 125–136 in Pik1 was not required for high affinity interaction with NCS-1. As observed for Frq1, Ca2+ binding did not appear to be required for stable association of NCS-1 with Pik1. As judged by a titration binding assay in which changes in intrinsic Trp fluorescence were used as a means to measure the amount of Pik1(120–192) required to saturate NCS-1, NCS-1 bound with an apparent KD of 150 nM, in good agreement with the measured affinity of Frq1-Pik1 complexes (Frq1-Pik1(110–192), apparent KD = 62 nM; Frq1-Pik1(151–192) apparent KD = 140 nM) (20). The observed Hill coefficient of 1 for the NCS-1-Pik1(120–192) interaction is also in agreement with the formation of the 1:1 Frq1-Pik1 complexes observed in yeast cell extracts (20).
As one approach to characterize the physicochemical nature of NCS-1 binding to Pik1, Ala and Lys scanning mutagenesis in the region of residues 147–171 was performed in the context of the Pik1(10–192) N-terminal fragment. None of the Ala substitution mutants tested (18/39) was able to significantly alter NCS-1 binding, as judged by the in vitro pull-down assay, whereas at four positions where a change to Ala had no effect, substitution of Lys either modestly (S164K and I170K) or drastically (L160K and A171K) reduced NCS-1 binding. The four mutations that affected NCS-1 binding are located in a very hydrophobic segment of the Pik1 N terminus, 156VAPALVLSSMIMSAIAFP173, comprising exclusively non-polar and uncharged residues. Thus, our results indicate that binding of Pik1 to NCS-1 is mediated primarily by hydrophobic contacts, as observed for the interaction of Frq1 with Pik1 (20).
The crystal structure of Ca2+-saturated NCS-1 (2) and the NMR-derived model of the structure of Ca2+-bound Frq1 (3) revealed that both proteins have a wide solvent-exposed crevice, lined predominantly with hydrophobic residues, a feature that is not present in the structures of other members of the family of small, N-myristoylated, Ca2+-binding proteins, such as neurocalcin or recoverin (6, 8, 57). This cleft is the most likely structural element to provide the hydrophobic environment necessary for docking of the hydrophobic sequence element in Pik1 that constitutes its Frq1- (and NCS-1-) binding site. The spacing of the residues (Leu160, Ser164, Ile170, Ala171) where a change to Lys had some effect on NCS-1 binding is reminiscent of how they would be situated if they were located on one side of an -helix. Indeed, preliminary HSQC spectra for [15N]Pik1(110–192) bound to Ca2+-saturated Frq1 show that both segments which make contact with Frq1 (Phe125–Gln136 and Ala157–Ser169, respectively) also assume an -helical conformation in the complex (20).3 Hence, interaction of Pik1 with either Frq1 or NCS-1 is based on a very similar, if not identical, mechanism and involves the same binding domains. Presence of the hydrophobic crevice, which, so far, seems unique to frequenins among the members of the family of small, N-myristoylated, Ca2+-binding proteins for which structures have been determined at atomic resolution, could help explain how frequenin can interact with a physiological target(s) that does not overlap with the targets of other members of the family in the same cell type. If so, the fact that KChIP2 was unable to interact with Pik1 predicts that it lacks such a hydrophobic cleft.
Given that prior work has established that the only essential role of Frq1 in yeast cells is association with and regulation of Pik1, our findings demonstrate that NCS-1 is also capable of fulfilling these functions, at least in yeast. It remains to be seen, however, whether NCS-1 directly interacts with and regulates the function and/or localization of a vertebrate PtdIns 4-kinase. If so, then frequenins, in general, might serve as regulators of PtdIns 4-kinases in all cell types.
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FOOTNOTES |
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These authors contributed equally to this work.
|| To whom correspondence should be addressed. Tel.: 49-40-428-03-5081; Fax: 49-40-428-03-5102; E-mail: pointuri{at}uke.uni-hamburg.de.
1 The abbreviations used are: GCAP, guanylyl cyclase-activating protein; KChIP, potassium channel-interacting protein; DTT, dithiothreitol; NTA, nitrilotriacetic acid; NCS-1, neuronal calcium sensor-1; PtdIns, phosphatidylinositol; PtdIns4K, phosphatidylinositol 4-kinase ; AD, activation domain; BD, DNA-binding domain; HSQC, heteronuclear single quantum coherence.
2 The anti-NCS-1 antibodies we used were raised in rabbits against bacterially expressed and thus unmyristoylated NCS-1. Therefore, an alternative explanation for the apparent expression pattern observed, which we cannot rule out at this time, is that a significant fraction of the IgG in the anti-NCS-1 antiserum is directed against the N terminus of NCS-1 and, when the protein is N-myristoylated, it reacts less well with the antiserum.
3 J. B. Ames, personal communication.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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nm, wavelength of emission spectrum. B, saturation binding curve for the interaction of NCS-1 with Pik1(120–192)-His6. Binding data were obtained from fluorescence intensity measurements at 340 nm (