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Functional analysis of calcium-binding EF-hand motifs of visinin-like protein-1
BBRC Biochemical and Biophysical Research Communications 296 (2002) 827–832 www.academicpress.com
Functional analysis of calcium-binding EF-hand motifs of visinin-like protein-1 Lin Lin,a Karl-Heinz Braunewell,b,c Eckart D. Gundelfinger,b and Rene Ananda,d,* a
Neuroscience Center of Excellence, 2020 Gravier Street, Suite D, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA b Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, P.O. Box 1860, Magdeburg D-39008, Germany c Signal Transduction Research Group, Neuroscience Research Center of the Charite, Humboldt University, Berlin, Germany d Department of Neurology, 2020 Gravier Street, Suite D, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA Received 23 July 2002
Abstract Visinin-like protein-1 (VILIP-1), a myristoylated calcium sensor protein with three EF-hand motifs, modulates adenylyl cyclase activity. It translocates to membranes when a postulated ‘‘calcium-myristoyl switch’’ is triggered by calcium-binding to expose its sequestered myristoyl moiety. We investigated the contributions of the EF-hand motifs to the translocation of VILIP-1 to membranes and to the modulation of adenylyl cyclase activity. Mutation of residues crucial for binding calcium within each one of the EF-hand motifs indicated that they all contributed to binding calcium. Simultaneous mutations of all of the three EF-hand motifs completely abolished VILIP-1’s ability to bind calcium, attenuated but did not eliminate its modulation of adenylyl cyclase activity, and abolished its calcium-dependence for association with cellular membranes. These results show that the calcium-binding EFhand motifs of VILIP-1 do not have an essential role in modulating adenylyl cyclase activity but instead have a structural role in activating the ‘‘calcium-myristoyl switch’’ of VILIP-1. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Visinin-like protein-1; EF-hand; Calcium; Cyclic AMP; Adenylyl cyclase; Myristoylation
Visinin-like protein-1 (VILIP-1) is a member of a large family of neuronal calcium sensor proteins that has been classified into five subfamilies termed groups I–V (reviewed in [1]). VILIP-1 belongs to the group III family, which consists of VILIP-1, VILIP-2, and VILIP3 [2–7]. VILIP-1 has four EF-hand motifs of which only EF-hand 2, 3, and 4 are thought to be functional because EF-hand 1 lacks two oxygen-containing side chain residues crucial for binding calcium. The EF-hand motif is a highly conserved calcium-binding motif found in a large number of intracellular proteins. The term EF-hand was first used in describing the structure of parvalbumin [8]. In an EF-hand, the calcium ion is coordinated in a pentagonal–bipyramidal configuration. The calcium ion is coordinated by side chain oxygen atoms of residues at positions X, Y, and Z, by the backbone carbonyl of residue at position-Y, by a water
*
Corresponding author. Fax: 1-504-599-0891. E-mail address: [email protected] (R. Anand).
molecule held in position by the residue at position-X, and by an acidic side chain of the residue at position-Z. VILIP-1 is myristoylated on a glycine residue at the second position on its polypeptide chain. The myristolylation of this residue presumably facilitates its targeting to membranes. Interestingly, within most, but not all members of this calcium sensor protein family, the myristoyl group that is sequestered is exposed for targeting to the membrane after these proteins bind calcium [9,10]. This conformational change has been termed the ‘‘calcium-myristoyl switch.’’ Members of the VILIP-1 family act as calcium sensors that modulate intracellular signaling pathways by directly or indirectly regulating the activity of adenylyl cyclase [11]. In this paper, we used a site-directed mutagenesis strategy to systematically investigate the contributions of each of the three putative functional EF-hand motifs 2, 3, and 4 to the functional properties of VILIP-1. We found that all three EF-hand motifs contributed to binding calcium. Mutation of EF-hand motifs 2, 3, and 4 of VILIP-1 abolished its calcium-
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 9 4 3 - 9
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dependence for binding membranes and attenuated but did not abolish its ability to modulate adenylyl cyclase activity. In contrast, the myristoyl moiety was absolutely required for is ability to associate with membranes and modulate adenylyl cyclase activity. These results suggest that the calcium-binding EF-hand motifs of VILIP-1 have a structural role in triggering the ‘‘calcium-myristoyl switch’’ of VILIP-1 but are not essential for modulation of adenylyl cyclase activity. Materials and methods Constructs. All constructs were made by the polymerase chain reaction (PCR) using appropriate pairs of forward and reverse synthetic oligonucleotide primers (Life Technologies, Bethesda, MD) and Pfu Turbo (Stratagene, San Diego, CA). All DNA sequence analysis was done using the ThermoSequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech, Piscataway, NJ). For all primers, the restriction enzyme sites are underlined. The wild-type VILIP-1 cDNA was amplified from mouse total brain cDNAs using the forward primer 50 -GGG GAT CCG CCA CCA TGG GGA AAC AGA ATA GCA AA and the reverse primer 50 -GGG TCT AGA TCA TTT CTG AAT GTC ACA CTG CAG and the PCR amplified cDNA ligated into the BamHI–XbaI sites of the mammalian cell expression vector pEF6/myc-HisA (Invitrogen, Carlsbad, CA). The expressed protein lacked the myc-his tag because of the presence of the endogeneous stop codon present in each cloned cDNA. Mutagenesis was performed using the Quik-Change site-directed mutagenesis kit (Stratagene, San Diego, CA) using the following primers 50 -GAT GGC ACC ATC GTT TTC CGA GAG TTC and 50 -GAA CTC TCG GAA AAC GAT GGT GCC ATC to mutagenize residue Asp81 to Val in the EF-hand motif 2 (mVILIP-X2EF ); 50 -GGT GAC GGC AAG ATC GCC CGA GTG GAG ATG CTG G and 50 -CCA GCA TCT CCA CTC GGG CGA TCT TGC CGT CAC C to mutagenize residue Thr117 to Ala in the EF-hand motif 3 (mVILIP-X3EF ); and 50 -GAA CAA AGA TGA CCA GAT TGC ACT GGA TGA ATT CAA AGA AGC TGC and 50 -GCA GCT TCT TTG AAT TCA TCC AGT GCA ATC TGG TCA TCT TTG TTC to mutagenize residue Thr167 to Ala in EF-hand motif 4 (mVILIP-X4EF ). The triple mutant (mVILIPX2;3;4EF ) was generated by sequential mutagenesis using the same sets of primers. The non-myristoylatable mVILIP-1myr mutant was generated by mutagenesis of residue glycine 2 to alanine using the primers: 50 -GGA TCC GCC ACC ATG GCG AAA CAG AAT AGC AAA CTG G and 50 -CCA GTT TGC TAT TCT GTT TCG CCA TGG TGG CGG ATC C. Antibodies. The anti-VILIP-1 rabbit serum used for the immunoblots has been previously described [13] and the goat anti-rabbit horseradish peroxidase conjugated antibodies (Abs) were obtained from Pierce, Rockford, IL. The Alexa Fluor 546-conjugated goat antirabbit secondary Ab was obtained from Molecular Probes, Eugene, OR and used at a 1/1000 dilution. Expression of recombinant VILIP in tsA 201 cells. tsA 201 cells (derived from human kidney embryonic (HEK) cells) were cultured in 6-well plates in DMEM (Life Technologies, Bethesda, MD) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 lg=ml streptomycin. Cells were transfected at 90% confluency (106 cells=well) with various cDNAs cloned into the vector pEF6/myc-His A (Invitrogen, Carlsbad, CA) using lipofectamine 2000 (Life Technologies, Bethesda, MD) as per manufacturer’s instructions and used after 24–36 h. Mobility shift assay. Cells expressing recombinant wild-type and mutant VILIP-1 proteins were homogenized in 1 ml buffer containing 5 mM Tris–HCl (pH7.5), 0.1 M NaCl, and 1 mM PMSF supplemented with either 2 mM EGTA or 1 mM CaCl2 . The homogenate was centrifuged at 15,000g for 20 min and the supernatants were subjected to
immunoblot analysis. Protein concentrations were measured using the Bio-Rad detergent compatible (DC) protein assay. Western blot analysis. Samples were electrophoresed in 12% SDS– polyacrylamide gels and electrotransferred onto PVDF membrane (IMMUN-BLOT; Bio-Rad Laboratories). Blots were blocked with PBS containing 0.1% Tween 20 and 5% fat-free milk powder for 1 h at room temperature and then incubated overnight at 4 °C with the primary Ab (1:5000 dilution). The immunoreactivity was detected with the Super Signal Chemiluminescent-HRP substrate system (SuperSignal, Pierce, Rockford, IL). Cytosolic and membrane protein distribution assay. tsA 201 cells were harvested 24 h after transfection and then the cells were sonicated in 500 ll buffer containing 5 mM Tris–HCl (pH 7.5), 0.1 M NaCl, and 1 mM PMSF supplemented either with 2 mM EGTA or 1 mM CaCl2 . The homogenate was centrifuged at 15,000g for 20 min and the supernatant was considered the soluble protein fraction. The membrane pellet was re-sonicated in 500 ll buffer containing 2% NP-40, 2 mM EGTA or 1 mM CaCl2 , agitated for 2 h at 4 °C, centrifuged at 15,000g for 20 min and the supernatant was considered the membrane protein fraction. Protein concentrations were determined using the Bio-Rad Detergent Compatible (DC) Protein Assay. Immunocytochemistry. tsA 201 cells transfected with the various VILIP-1 cDNAs were treated with or without A23187 (1 lM, 15 min) and then fixed with 3% formaldehyde in PBS for 5 min at room temperature, washed for 5 min intervals three times with PBS, and then used for immunocytochemical staining. Cells were blocked with PBS containing 0.5% Triton X-100 and 2% bovine serum albumin for 1 h and then incubated with the primary VILIP-1 Ab (1:1000) diluted in the same solution containing 4% normal goat serum at 4 °C overnight. Cells were washed for 15 min intervals three times and then incubated with the Alexa Fluor 546-conjugated goat anti-rabbit Ab for 1 h at room temperature. After three washes, these cells were visualized by fluorescence microscopy using an Olympus BX50i upright fluorescence microscope through a 60 water-immersion lens. Images were acquired through an OLY750 digital three-chip color camera with the Image Pro Plus software (Media, Silver Springs, MD). Intracellular cyclic AMP assay. Thirty-six hours after transfection, tsA 201 cells were incubated with 3-iso-butyl-1-methylxanthine (IBMX; 1 mM) in DMEM medium for 2 h at 37 °C in a 5% CO2 incubator. The medium was aspirated and the cells were extracted with 1 ml ice-cold 65% ethanol at )20 °C overnight. The extracts were centrifuged at 2000 rpm for 15 min and the clear supernatant was transferred to fresh tubes and evaporated in a vacuum oven, following which the dried extracts were dissolved in 50 ll distilled H2 O. cAMP levels were measured using the Cyclic AMP ð3 HÞ Assay System (Amersham Pharmacia Biotech, Piscataway, NJ).
Results All three functional EF-hand motifs 2, 3, and 4 contribute to the calcium-induced conformational changes of VILIP-1 The EF-hand structure consists of two a-helical segments bridged by a calcium-binding loop. The loop is formed by 12 amino acid stretches, six of which (designated as X, Y, Z, Y , X , Z) participate in the coordination of one calcium ion. Only EF-hand motifs 2, 3, and 4 of VILIP-1 have the canonical motif that is required to bind calcium. We examined the contributions of each of these EF-hand motifs to the ‘‘calcium-myristoyl switch’’ and the functional properties of VILIP-1, by mutating the residue at the X position within each of the three putative calcium-binding EF-hand motifs. We
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generated mutants mVILIP2EF (Asp81Val), mVILIPX3EF (Thr117Ala), mVILIP-X4EF (Thr167Ala), the double mutant mVILIP-X3;4EF , and the triple mutant mVILIP-X2;3;4EF . We monitored the calcium-induced shift in their mobility on SDS–PAGE gels in the presence of calcium (1 mM) versus the absence of calcium (due to the presence of the chelator EGTA (2 mM)). This mobility shift has been previously demonstrated to correlate with VILIP-1’s ability to bind calcium and undergo a calciuminduced conformational change [12]. All three mutants harboring mutations within each of the three functional EF-hand motifs 2, 3, and 4 showed altered mobility shifts in the presence of calcium like VILIP-1 but to different degrees (Fig. 1A). By comparing the relative mobility shift of each of the mutants in the presence or absence of calcium, we demonstrate that the mobility shift was not due to the mutations themselves but due to the loss of calcium-binding (Fig. 1B). The stepwise shift in mobility for each of the mutants mVILIP-X2EF , mVILIP-X3EF , and mVILIP-X4EF suggested that all three EF-hand motifs were capable of binding calcium and contributed to the calcium-induced conformational changes observed for VILIP-1. The mVILIP-X2;3;4EF mutant harboring mutations within all three of its EF-hand motifs showed virtually no shift in mobility in the presence of calcium compared to the
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absence of calcium in keeping with the prediction that only EF-hand motifs 2, 3, and 4 and not EF-hand motif 1 are capable of binding calcium. A second triple mutant, mVILIP-Z2;3;4EF , in which the acidic side chain residue at position Z was also mutated to alanine in all three EF-hand motifs, also gave identical results to those obtained with mVILIP-X2;3;4EF . Calcium-independent association of mVILIP-X 2;3;4EF and mVILIP-Z 2;3;4EF with membranes VILIP-1 has been reported to preferentially associate with cellular membranes in the presence of calcium. To determine if the calcium-binding EF-hand motifs were essential for this association, we measured the relative distribution of VILIP-1, mVILIP-X2;3;4EF , and mVILIPZ2;3;4EF in the cytosolic (C) fraction versus the membrane (M) fraction in the presence of high calcium (1 mM) or low calcium (2 mM EGTA). VILIP-1 showed a preferential association with membranes in the presence of calcium than in the absence of calcium. mVILIP-X2;3;4EF and mVILIP-Z2;3;4EF , on the other hand, were almost exclusively associated with membranes both in the absence and presence of calcium. Myristoylation of VILIP-1 is crucial for its functional properties A glycine residue at the second position of the polypeptide chain of VILIP-1 is myristoylated. To confirm that myristolylation of VILIP-1 is important for its ability to associate with cellular membranes in a calcium-dependent manner and to modulate adenylyl cyclase activity in tsA 201 cells, as previously demonstrated in transfected C6 glioma cells, the glycine residue of VILIP-1 at the second position of its polypeptide was mutated to alanine and the resulting mutant was designated as mVILIPmyr . mVILIPmyr showed a slight difference in the mobility shift compared to the wild-type VILIP-1 in the presence of calcium, suggesting that the mutation partially altered its ability to conformationally respond to calcium (Figs. 1A and B). mVILIPmyr failed to show an increased membranous distribution in the presence of calcium (Fig. 2) and its ability to modulate adenylyl cyclase activity was completely abolished (Fig. 4). The myristoyl moiety was found to be a crucial mediator of VILIP-1’s ability to bind membranes in the presence of calcium and functionally modulate adenylyl cyclase activity in tsA 201 cells, consistent with results previously reported for VILIP-1 in the C6 glioma cells.
Fig. 1. Calcium-induced mobility shift. Immunoblot analysis of mobility shift of wild-type and mutant VILIP-1 proteins in the presence (+) or absence ()) of calcium. (A) Relative mobility shift of mutant VILIP-1s compared to wild-type VILIP-1; (B) relative mobility shift of VILIP-1 and VILIP-1 mutants in the presence (+) or absence ()) of calcium.
Cellular distribution of recombinant wild-type and mutant VILIP-1 in tsA201 cells To determine if the calcium-dependent changes in the distribution of VILIP-1, mVILIPmyr , mVILIP-X2;3;4EF ,
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Fig. 2. Calcium-induced change in membrane versus cytosolic distribution. Equivalent amounts of cytosolic proteins (C) and membrane proteins (M), from tsA 201 cells expressing the various VILIP-1s, were isolated in the presence or absence of calcium and separated by SDS– PAGE. The relative distribution of VILIP-1 was then detected by immunoblot analysis.
and mVILIP-Z2;3;4EF between the cytosol and the membrane in a cellular environment were similar to those determined by the subcellular fractionation experiments, their distribution in transfected tsA 201 cells was monitored by immunofluorescence microscopy. Cells expressing the VILIP-1 proteins were treated with or without the calcium ionophore A23187 (1lM, 15 min) and the distribution of the VILIP-1 proteins was visualized by staining fixed cells with an anti-VILIP-1 Ab. The staining was visualized in conjunction with an Alexa Fluor 546-conjugated secondary Ab. The membrane versus cytosolic distribution obtained in all cases (Fig. 3), in the presence or absence of calcium, by the immunostaining experiments were found to be similar to those obtained with the subcellular fractionation method.
Fig. 3. Immunocytochemical detection of membranous and cytosolic VILIP-1 in tsA 201 cells. tsA 201 cells transfected with various VILIP-1 cDNAs were treated with or without A23187 (1 lM, 15 min), immunostained, and visualized by immunofluorescence microscopy.
mVILIP-X4EF , mVILIP-X3;4EF , mVILIP-X2;3;4EF , and mVILIP-Z2;3;4EF . An increase in the cellular levels of cAMP was observed for all three single mutants (data not shown). The triple mutants mVILIP-X2;3;4EF and mVILIP-Z2;3;4EF retained their ability to modulate adenylyl cyclase activity but with significantly reduced efficacy (Fig. 4).
EF-hand motifs 2, 3, and 4 are not essential for modulation of adenylyl cyclase activity Discussion Expression of VILIP-1 in glioma C6 cells increases cellular levels of cAMP [13]. We measured the ability of VILIP-1 to modulate adenylyl cyclase activity by comparing the cellular levels of cAMP in tsA 201 cells transfected with VILIP-1 cDNA versus those transfected with the vector plasmid alone, in the presence of the cAMP phosphodiesterase inhibitor IBMX (1 mM). VILIP-1 increased the cellular levels of cAMP 2-fold during a 2 h period over those transfected with vector alone only in the presence of IBMX. No change in cAMP was observed in cells transfected with VILIP-1 over the same time period in the absence of IBMX (data not shown). This result ruled out the possibility that VILIP-1 increases the cellular levels of cAMP by acting as a cAMP phosphodiesterase inhibitor. We used this change in cellular levels of cAMP as one measure of VILIP-1’s functionality. We next measured cAMP levels in cells transfected with mVILIP-X2EF , mVILIP-X3EF ,
VILIP-1 is a calcium sensor protein that is known to bind calcium, associate with membranes in a calciumdependent manner, and modulate adenylyl cyclase activity, in response to changes in cellular levels of calcium. VILIP-1 has been postulated to undergo a conformational change that unmasks a sequestered myristoyl moiety after it binds calcium, and thus, associates with membranes to directly or indirectly activate adenylyl cyclase [13]. To elucidate the contributions of its EF-hand motifs and myristoyl moiety to its functionality, we systematically mutated the three EF-hand motifs 2, 3, and 4, and the myristoylated residue of VILIP-1 and studied the functional properties of the resulting VILIP-1 mutants. All three EF-hand motifs 2, 3, and 4 were found to contribute to inducing calcium-induced conformational changes in VILIP-1. Mutation of all three EF-hands 2,
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have recently found that VILIP-1 interacts with the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) a4 subunit and modulates AChR functional activity [24]. Because AChRs show significant permeability to calcium, it is likely that the EF-hand motifs of VILIP-1 respond to the calcium influx through these channels to trigger the ‘‘calcium-myristoyl switch,’’ and thus, translocate VILIP-1 to the cell surface membrane where it interacts with AChRs.
Acknowledgments Research in the laboratory of R.A. is supported by grants from the National Science Foundation (NSF/LEQSF(2001-04)-R-II-01) and the Millennium Trust Health Excellence Fund HEF (2002–2007)-SCP-01. Research in the laboratory of K.-H.B. and E.D.G. is supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. Thanks to Ms. Magdalen Treuil (supported with funds from the LSUHSC Parkinson’s Research Fund) for the excellent technical support. Fig. 4. Modulation of adenylyl cyclase activity. cAMP levels were measured from tsA 201 cells expressing the various VILIP-1s. Control cells were those transfected with the vector pEF6/myc-His A alone. The values shown are normalized to the value obtained from the control and represent means SE of three independent experiments carried out in duplicate.
3, and 4 reduced but did not abolish VILIP-1’s ability to modulate adenylyl cyclase activity. In addition, these mutations significantly increased its association with cellular membranes. Because mVILIP-X2;3;4EF and mVILIP-Z2;3;4EF mutants failed to exhibit any calciuminduced conformational changes, as measured by the mobility shift assays, their increased association with membranes suggested that the myristoyl moiety, that mediates association with membranes, was constitutively exposed. Interestingly, the ‘‘calcium-myristoyl switch’’ operates in reverse for another myristoylated calcium sensor protein called guanylyl cyclase activating protein-2, because its disassociation from membranes at high concentrations of calcium was observed [14]. All three functional EF-hand motifs of guanylyl cyclase activating protein-2 were found to contribute to the inhibitory effects of calcium on its ability to activate guanylyl cyclase [15]. Several recent reports support a role for calcium sensor proteins in coupling changes in intracellular levels of calcium to modulation of different types of membrane proteins including voltage- and ligand-gated channels. For example, the calcium sensor proteins KChIP [16], frequenin [17], and NCS-1 [18] were shown to modulate the A-type Kþ channels and NCS-1 was shown to modulate the voltage-gated calcium channels [19]. Calmodulin also has a well-established role in regulating the functional properties of NMDA receptors [20,21] and voltage-gated calcium channels [22,23]. We
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Functional analysis of calcium-binding EF-hand motifs of visinin-like protein-1 Lin Lin,a Karl-Heinz Braunewell,b,c Eckart D. Gundelfinger,b and Rene Ananda,d,* a
Neuroscience Center of Excellence, 2020 Gravier Street, Suite D, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA b Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, P.O. Box 1860, Magdeburg D-39008, Germany c Signal Transduction Research Group, Neuroscience Research Center of the Charite, Humboldt University, Berlin, Germany d Department of Neurology, 2020 Gravier Street, Suite D, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA Received 23 July 2002
Abstract Visinin-like protein-1 (VILIP-1), a myristoylated calcium sensor protein with three EF-hand motifs, modulates adenylyl cyclase activity. It translocates to membranes when a postulated ‘‘calcium-myristoyl switch’’ is triggered by calcium-binding to expose its sequestered myristoyl moiety. We investigated the contributions of the EF-hand motifs to the translocation of VILIP-1 to membranes and to the modulation of adenylyl cyclase activity. Mutation of residues crucial for binding calcium within each one of the EF-hand motifs indicated that they all contributed to binding calcium. Simultaneous mutations of all of the three EF-hand motifs completely abolished VILIP-1’s ability to bind calcium, attenuated but did not eliminate its modulation of adenylyl cyclase activity, and abolished its calcium-dependence for association with cellular membranes. These results show that the calcium-binding EFhand motifs of VILIP-1 do not have an essential role in modulating adenylyl cyclase activity but instead have a structural role in activating the ‘‘calcium-myristoyl switch’’ of VILIP-1. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Visinin-like protein-1; EF-hand; Calcium; Cyclic AMP; Adenylyl cyclase; Myristoylation
Visinin-like protein-1 (VILIP-1) is a member of a large family of neuronal calcium sensor proteins that has been classified into five subfamilies termed groups I–V (reviewed in [1]). VILIP-1 belongs to the group III family, which consists of VILIP-1, VILIP-2, and VILIP3 [2–7]. VILIP-1 has four EF-hand motifs of which only EF-hand 2, 3, and 4 are thought to be functional because EF-hand 1 lacks two oxygen-containing side chain residues crucial for binding calcium. The EF-hand motif is a highly conserved calcium-binding motif found in a large number of intracellular proteins. The term EF-hand was first used in describing the structure of parvalbumin [8]. In an EF-hand, the calcium ion is coordinated in a pentagonal–bipyramidal configuration. The calcium ion is coordinated by side chain oxygen atoms of residues at positions X, Y, and Z, by the backbone carbonyl of residue at position-Y, by a water
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Corresponding author. Fax: 1-504-599-0891. E-mail address: [email protected] (R. Anand).
molecule held in position by the residue at position-X, and by an acidic side chain of the residue at position-Z. VILIP-1 is myristoylated on a glycine residue at the second position on its polypeptide chain. The myristolylation of this residue presumably facilitates its targeting to membranes. Interestingly, within most, but not all members of this calcium sensor protein family, the myristoyl group that is sequestered is exposed for targeting to the membrane after these proteins bind calcium [9,10]. This conformational change has been termed the ‘‘calcium-myristoyl switch.’’ Members of the VILIP-1 family act as calcium sensors that modulate intracellular signaling pathways by directly or indirectly regulating the activity of adenylyl cyclase [11]. In this paper, we used a site-directed mutagenesis strategy to systematically investigate the contributions of each of the three putative functional EF-hand motifs 2, 3, and 4 to the functional properties of VILIP-1. We found that all three EF-hand motifs contributed to binding calcium. Mutation of EF-hand motifs 2, 3, and 4 of VILIP-1 abolished its calcium-
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dependence for binding membranes and attenuated but did not abolish its ability to modulate adenylyl cyclase activity. In contrast, the myristoyl moiety was absolutely required for is ability to associate with membranes and modulate adenylyl cyclase activity. These results suggest that the calcium-binding EF-hand motifs of VILIP-1 have a structural role in triggering the ‘‘calcium-myristoyl switch’’ of VILIP-1 but are not essential for modulation of adenylyl cyclase activity. Materials and methods Constructs. All constructs were made by the polymerase chain reaction (PCR) using appropriate pairs of forward and reverse synthetic oligonucleotide primers (Life Technologies, Bethesda, MD) and Pfu Turbo (Stratagene, San Diego, CA). All DNA sequence analysis was done using the ThermoSequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech, Piscataway, NJ). For all primers, the restriction enzyme sites are underlined. The wild-type VILIP-1 cDNA was amplified from mouse total brain cDNAs using the forward primer 50 -GGG GAT CCG CCA CCA TGG GGA AAC AGA ATA GCA AA and the reverse primer 50 -GGG TCT AGA TCA TTT CTG AAT GTC ACA CTG CAG and the PCR amplified cDNA ligated into the BamHI–XbaI sites of the mammalian cell expression vector pEF6/myc-HisA (Invitrogen, Carlsbad, CA). The expressed protein lacked the myc-his tag because of the presence of the endogeneous stop codon present in each cloned cDNA. Mutagenesis was performed using the Quik-Change site-directed mutagenesis kit (Stratagene, San Diego, CA) using the following primers 50 -GAT GGC ACC ATC GTT TTC CGA GAG TTC and 50 -GAA CTC TCG GAA AAC GAT GGT GCC ATC to mutagenize residue Asp81 to Val in the EF-hand motif 2 (mVILIP-X2EF ); 50 -GGT GAC GGC AAG ATC GCC CGA GTG GAG ATG CTG G and 50 -CCA GCA TCT CCA CTC GGG CGA TCT TGC CGT CAC C to mutagenize residue Thr117 to Ala in the EF-hand motif 3 (mVILIP-X3EF ); and 50 -GAA CAA AGA TGA CCA GAT TGC ACT GGA TGA ATT CAA AGA AGC TGC and 50 -GCA GCT TCT TTG AAT TCA TCC AGT GCA ATC TGG TCA TCT TTG TTC to mutagenize residue Thr167 to Ala in EF-hand motif 4 (mVILIP-X4EF ). The triple mutant (mVILIPX2;3;4EF ) was generated by sequential mutagenesis using the same sets of primers. The non-myristoylatable mVILIP-1myr mutant was generated by mutagenesis of residue glycine 2 to alanine using the primers: 50 -GGA TCC GCC ACC ATG GCG AAA CAG AAT AGC AAA CTG G and 50 -CCA GTT TGC TAT TCT GTT TCG CCA TGG TGG CGG ATC C. Antibodies. The anti-VILIP-1 rabbit serum used for the immunoblots has been previously described [13] and the goat anti-rabbit horseradish peroxidase conjugated antibodies (Abs) were obtained from Pierce, Rockford, IL. The Alexa Fluor 546-conjugated goat antirabbit secondary Ab was obtained from Molecular Probes, Eugene, OR and used at a 1/1000 dilution. Expression of recombinant VILIP in tsA 201 cells. tsA 201 cells (derived from human kidney embryonic (HEK) cells) were cultured in 6-well plates in DMEM (Life Technologies, Bethesda, MD) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 lg=ml streptomycin. Cells were transfected at 90% confluency (106 cells=well) with various cDNAs cloned into the vector pEF6/myc-His A (Invitrogen, Carlsbad, CA) using lipofectamine 2000 (Life Technologies, Bethesda, MD) as per manufacturer’s instructions and used after 24–36 h. Mobility shift assay. Cells expressing recombinant wild-type and mutant VILIP-1 proteins were homogenized in 1 ml buffer containing 5 mM Tris–HCl (pH7.5), 0.1 M NaCl, and 1 mM PMSF supplemented with either 2 mM EGTA or 1 mM CaCl2 . The homogenate was centrifuged at 15,000g for 20 min and the supernatants were subjected to
immunoblot analysis. Protein concentrations were measured using the Bio-Rad detergent compatible (DC) protein assay. Western blot analysis. Samples were electrophoresed in 12% SDS– polyacrylamide gels and electrotransferred onto PVDF membrane (IMMUN-BLOT; Bio-Rad Laboratories). Blots were blocked with PBS containing 0.1% Tween 20 and 5% fat-free milk powder for 1 h at room temperature and then incubated overnight at 4 °C with the primary Ab (1:5000 dilution). The immunoreactivity was detected with the Super Signal Chemiluminescent-HRP substrate system (SuperSignal, Pierce, Rockford, IL). Cytosolic and membrane protein distribution assay. tsA 201 cells were harvested 24 h after transfection and then the cells were sonicated in 500 ll buffer containing 5 mM Tris–HCl (pH 7.5), 0.1 M NaCl, and 1 mM PMSF supplemented either with 2 mM EGTA or 1 mM CaCl2 . The homogenate was centrifuged at 15,000g for 20 min and the supernatant was considered the soluble protein fraction. The membrane pellet was re-sonicated in 500 ll buffer containing 2% NP-40, 2 mM EGTA or 1 mM CaCl2 , agitated for 2 h at 4 °C, centrifuged at 15,000g for 20 min and the supernatant was considered the membrane protein fraction. Protein concentrations were determined using the Bio-Rad Detergent Compatible (DC) Protein Assay. Immunocytochemistry. tsA 201 cells transfected with the various VILIP-1 cDNAs were treated with or without A23187 (1 lM, 15 min) and then fixed with 3% formaldehyde in PBS for 5 min at room temperature, washed for 5 min intervals three times with PBS, and then used for immunocytochemical staining. Cells were blocked with PBS containing 0.5% Triton X-100 and 2% bovine serum albumin for 1 h and then incubated with the primary VILIP-1 Ab (1:1000) diluted in the same solution containing 4% normal goat serum at 4 °C overnight. Cells were washed for 15 min intervals three times and then incubated with the Alexa Fluor 546-conjugated goat anti-rabbit Ab for 1 h at room temperature. After three washes, these cells were visualized by fluorescence microscopy using an Olympus BX50i upright fluorescence microscope through a 60 water-immersion lens. Images were acquired through an OLY750 digital three-chip color camera with the Image Pro Plus software (Media, Silver Springs, MD). Intracellular cyclic AMP assay. Thirty-six hours after transfection, tsA 201 cells were incubated with 3-iso-butyl-1-methylxanthine (IBMX; 1 mM) in DMEM medium for 2 h at 37 °C in a 5% CO2 incubator. The medium was aspirated and the cells were extracted with 1 ml ice-cold 65% ethanol at )20 °C overnight. The extracts were centrifuged at 2000 rpm for 15 min and the clear supernatant was transferred to fresh tubes and evaporated in a vacuum oven, following which the dried extracts were dissolved in 50 ll distilled H2 O. cAMP levels were measured using the Cyclic AMP ð3 HÞ Assay System (Amersham Pharmacia Biotech, Piscataway, NJ).
Results All three functional EF-hand motifs 2, 3, and 4 contribute to the calcium-induced conformational changes of VILIP-1 The EF-hand structure consists of two a-helical segments bridged by a calcium-binding loop. The loop is formed by 12 amino acid stretches, six of which (designated as X, Y, Z, Y , X , Z) participate in the coordination of one calcium ion. Only EF-hand motifs 2, 3, and 4 of VILIP-1 have the canonical motif that is required to bind calcium. We examined the contributions of each of these EF-hand motifs to the ‘‘calcium-myristoyl switch’’ and the functional properties of VILIP-1, by mutating the residue at the X position within each of the three putative calcium-binding EF-hand motifs. We
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generated mutants mVILIP2EF (Asp81Val), mVILIPX3EF (Thr117Ala), mVILIP-X4EF (Thr167Ala), the double mutant mVILIP-X3;4EF , and the triple mutant mVILIP-X2;3;4EF . We monitored the calcium-induced shift in their mobility on SDS–PAGE gels in the presence of calcium (1 mM) versus the absence of calcium (due to the presence of the chelator EGTA (2 mM)). This mobility shift has been previously demonstrated to correlate with VILIP-1’s ability to bind calcium and undergo a calciuminduced conformational change [12]. All three mutants harboring mutations within each of the three functional EF-hand motifs 2, 3, and 4 showed altered mobility shifts in the presence of calcium like VILIP-1 but to different degrees (Fig. 1A). By comparing the relative mobility shift of each of the mutants in the presence or absence of calcium, we demonstrate that the mobility shift was not due to the mutations themselves but due to the loss of calcium-binding (Fig. 1B). The stepwise shift in mobility for each of the mutants mVILIP-X2EF , mVILIP-X3EF , and mVILIP-X4EF suggested that all three EF-hand motifs were capable of binding calcium and contributed to the calcium-induced conformational changes observed for VILIP-1. The mVILIP-X2;3;4EF mutant harboring mutations within all three of its EF-hand motifs showed virtually no shift in mobility in the presence of calcium compared to the
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absence of calcium in keeping with the prediction that only EF-hand motifs 2, 3, and 4 and not EF-hand motif 1 are capable of binding calcium. A second triple mutant, mVILIP-Z2;3;4EF , in which the acidic side chain residue at position Z was also mutated to alanine in all three EF-hand motifs, also gave identical results to those obtained with mVILIP-X2;3;4EF . Calcium-independent association of mVILIP-X 2;3;4EF and mVILIP-Z 2;3;4EF with membranes VILIP-1 has been reported to preferentially associate with cellular membranes in the presence of calcium. To determine if the calcium-binding EF-hand motifs were essential for this association, we measured the relative distribution of VILIP-1, mVILIP-X2;3;4EF , and mVILIPZ2;3;4EF in the cytosolic (C) fraction versus the membrane (M) fraction in the presence of high calcium (1 mM) or low calcium (2 mM EGTA). VILIP-1 showed a preferential association with membranes in the presence of calcium than in the absence of calcium. mVILIP-X2;3;4EF and mVILIP-Z2;3;4EF , on the other hand, were almost exclusively associated with membranes both in the absence and presence of calcium. Myristoylation of VILIP-1 is crucial for its functional properties A glycine residue at the second position of the polypeptide chain of VILIP-1 is myristoylated. To confirm that myristolylation of VILIP-1 is important for its ability to associate with cellular membranes in a calcium-dependent manner and to modulate adenylyl cyclase activity in tsA 201 cells, as previously demonstrated in transfected C6 glioma cells, the glycine residue of VILIP-1 at the second position of its polypeptide was mutated to alanine and the resulting mutant was designated as mVILIPmyr . mVILIPmyr showed a slight difference in the mobility shift compared to the wild-type VILIP-1 in the presence of calcium, suggesting that the mutation partially altered its ability to conformationally respond to calcium (Figs. 1A and B). mVILIPmyr failed to show an increased membranous distribution in the presence of calcium (Fig. 2) and its ability to modulate adenylyl cyclase activity was completely abolished (Fig. 4). The myristoyl moiety was found to be a crucial mediator of VILIP-1’s ability to bind membranes in the presence of calcium and functionally modulate adenylyl cyclase activity in tsA 201 cells, consistent with results previously reported for VILIP-1 in the C6 glioma cells.
Fig. 1. Calcium-induced mobility shift. Immunoblot analysis of mobility shift of wild-type and mutant VILIP-1 proteins in the presence (+) or absence ()) of calcium. (A) Relative mobility shift of mutant VILIP-1s compared to wild-type VILIP-1; (B) relative mobility shift of VILIP-1 and VILIP-1 mutants in the presence (+) or absence ()) of calcium.
Cellular distribution of recombinant wild-type and mutant VILIP-1 in tsA201 cells To determine if the calcium-dependent changes in the distribution of VILIP-1, mVILIPmyr , mVILIP-X2;3;4EF ,
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Fig. 2. Calcium-induced change in membrane versus cytosolic distribution. Equivalent amounts of cytosolic proteins (C) and membrane proteins (M), from tsA 201 cells expressing the various VILIP-1s, were isolated in the presence or absence of calcium and separated by SDS– PAGE. The relative distribution of VILIP-1 was then detected by immunoblot analysis.
and mVILIP-Z2;3;4EF between the cytosol and the membrane in a cellular environment were similar to those determined by the subcellular fractionation experiments, their distribution in transfected tsA 201 cells was monitored by immunofluorescence microscopy. Cells expressing the VILIP-1 proteins were treated with or without the calcium ionophore A23187 (1lM, 15 min) and the distribution of the VILIP-1 proteins was visualized by staining fixed cells with an anti-VILIP-1 Ab. The staining was visualized in conjunction with an Alexa Fluor 546-conjugated secondary Ab. The membrane versus cytosolic distribution obtained in all cases (Fig. 3), in the presence or absence of calcium, by the immunostaining experiments were found to be similar to those obtained with the subcellular fractionation method.
Fig. 3. Immunocytochemical detection of membranous and cytosolic VILIP-1 in tsA 201 cells. tsA 201 cells transfected with various VILIP-1 cDNAs were treated with or without A23187 (1 lM, 15 min), immunostained, and visualized by immunofluorescence microscopy.
mVILIP-X4EF , mVILIP-X3;4EF , mVILIP-X2;3;4EF , and mVILIP-Z2;3;4EF . An increase in the cellular levels of cAMP was observed for all three single mutants (data not shown). The triple mutants mVILIP-X2;3;4EF and mVILIP-Z2;3;4EF retained their ability to modulate adenylyl cyclase activity but with significantly reduced efficacy (Fig. 4).
EF-hand motifs 2, 3, and 4 are not essential for modulation of adenylyl cyclase activity Discussion Expression of VILIP-1 in glioma C6 cells increases cellular levels of cAMP [13]. We measured the ability of VILIP-1 to modulate adenylyl cyclase activity by comparing the cellular levels of cAMP in tsA 201 cells transfected with VILIP-1 cDNA versus those transfected with the vector plasmid alone, in the presence of the cAMP phosphodiesterase inhibitor IBMX (1 mM). VILIP-1 increased the cellular levels of cAMP 2-fold during a 2 h period over those transfected with vector alone only in the presence of IBMX. No change in cAMP was observed in cells transfected with VILIP-1 over the same time period in the absence of IBMX (data not shown). This result ruled out the possibility that VILIP-1 increases the cellular levels of cAMP by acting as a cAMP phosphodiesterase inhibitor. We used this change in cellular levels of cAMP as one measure of VILIP-1’s functionality. We next measured cAMP levels in cells transfected with mVILIP-X2EF , mVILIP-X3EF ,
VILIP-1 is a calcium sensor protein that is known to bind calcium, associate with membranes in a calciumdependent manner, and modulate adenylyl cyclase activity, in response to changes in cellular levels of calcium. VILIP-1 has been postulated to undergo a conformational change that unmasks a sequestered myristoyl moiety after it binds calcium, and thus, associates with membranes to directly or indirectly activate adenylyl cyclase [13]. To elucidate the contributions of its EF-hand motifs and myristoyl moiety to its functionality, we systematically mutated the three EF-hand motifs 2, 3, and 4, and the myristoylated residue of VILIP-1 and studied the functional properties of the resulting VILIP-1 mutants. All three EF-hand motifs 2, 3, and 4 were found to contribute to inducing calcium-induced conformational changes in VILIP-1. Mutation of all three EF-hands 2,
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have recently found that VILIP-1 interacts with the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) a4 subunit and modulates AChR functional activity [24]. Because AChRs show significant permeability to calcium, it is likely that the EF-hand motifs of VILIP-1 respond to the calcium influx through these channels to trigger the ‘‘calcium-myristoyl switch,’’ and thus, translocate VILIP-1 to the cell surface membrane where it interacts with AChRs.
Acknowledgments Research in the laboratory of R.A. is supported by grants from the National Science Foundation (NSF/LEQSF(2001-04)-R-II-01) and the Millennium Trust Health Excellence Fund HEF (2002–2007)-SCP-01. Research in the laboratory of K.-H.B. and E.D.G. is supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. Thanks to Ms. Magdalen Treuil (supported with funds from the LSUHSC Parkinson’s Research Fund) for the excellent technical support. Fig. 4. Modulation of adenylyl cyclase activity. cAMP levels were measured from tsA 201 cells expressing the various VILIP-1s. Control cells were those transfected with the vector pEF6/myc-His A alone. The values shown are normalized to the value obtained from the control and represent means SE of three independent experiments carried out in duplicate.
3, and 4 reduced but did not abolish VILIP-1’s ability to modulate adenylyl cyclase activity. In addition, these mutations significantly increased its association with cellular membranes. Because mVILIP-X2;3;4EF and mVILIP-Z2;3;4EF mutants failed to exhibit any calciuminduced conformational changes, as measured by the mobility shift assays, their increased association with membranes suggested that the myristoyl moiety, that mediates association with membranes, was constitutively exposed. Interestingly, the ‘‘calcium-myristoyl switch’’ operates in reverse for another myristoylated calcium sensor protein called guanylyl cyclase activating protein-2, because its disassociation from membranes at high concentrations of calcium was observed [14]. All three functional EF-hand motifs of guanylyl cyclase activating protein-2 were found to contribute to the inhibitory effects of calcium on its ability to activate guanylyl cyclase [15]. Several recent reports support a role for calcium sensor proteins in coupling changes in intracellular levels of calcium to modulation of different types of membrane proteins including voltage- and ligand-gated channels. For example, the calcium sensor proteins KChIP [16], frequenin [17], and NCS-1 [18] were shown to modulate the A-type Kþ channels and NCS-1 was shown to modulate the voltage-gated calcium channels [19]. Calmodulin also has a well-established role in regulating the functional properties of NMDA receptors [20,21] and voltage-gated calcium channels [22,23]. We
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