From the Division of Molecular and Structural
Biology, Ontario Cancer Institute and Department of Medical
Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada and
the ¶ Institute of Medical Science, University of Toronto and
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
Ontario M5G 1X5, Canada
Received for publication, November 20, 2000, and in revised form, March 30, 2001
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ABSTRACT |
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Calexcitin (CE) is a calcium sensor protein that
has been implicated in associative learning. The CE gene was previously
cloned from the long-finned squid, Loligo pealei, and the
gene product was shown to bind GTP and modulate K+ channels
and ryanodine receptors in a Ca2+-dependent
manner. We cloned a new gene from L. pealei, which encodes
a CE-like protein, here named calexcitin B (CEB).
CEB has 95% amino acid identity to the original form. Our
sequence analyses indicate that CEs are homologous to the sarcoplasmic calcium-binding protein subfamily of the EF-hand superfamily. Far and
near UV circular dichroism and nuclear magnetic resonance studies
demonstrate that CEB binds Ca2+ and undergoes a
conformational change. CEB is phosphorylated by protein
kinase C, but not by casein kinase II. CEB does not bind
GTP. Western blot experiments using polyclonal antibodies generated
against CEB showed that CEB is expressed in the
L. pealei optic lobe. Taken together, the neuronal protein
CE represents the first example of a Ca2+ sensor in the
sarcoplasmic calcium-binding protein family.
Calcium ions (Ca2+) play a vital role in cells, being
involved in various signaling events from cell growth to cell death.
Many Ca2+-dependent cellular processes are
mediated by Ca2+-binding proteins
(CaBPs),1 which share a
common Ca2+ binding motif, termed "EF-hand" (1).
EF-hand proteins can be subdivided into two types. A
"Ca2+ sensor" regulates downstream target proteins in a
Ca2+-dependent manner and a "Ca2+
buffer" contributes to maintaining the intracellular Ca2+
level (2). Calmodulin, troponin C, S100 proteins, frequenin, and
neurocalcin are examples of Ca2+ sensors, whereas
sarcoplasmic calcium-binding proteins (SCPs), parvalbumin, and
calbindin D9k are thought to function as
Ca2+ buffers (2).
Recently, a new CaBP called calexcitin (CE) has been identified in
squid and implicated to play a role in associative learning through
inhibition of K+ channels in a
Ca2+-dependent manner (3-5). Subsequently, it
was reported (6) that CE binds and activates ryanodine receptors, which
are also involved in associative learning (7). CE has been shown to possess GTP binding and GTPase activities (4), but its functional significance is unknown. The gene encoding CE was cloned, and the
recombinant protein was shown to bind Ca2+ and to be a high
affinity substrate for protein kinase C (PKC) (4). To date, in addition
to ryanodine receptor activation (6) and K+ channel
inhibition (4), a number of other functions have been proposed for CE,
including an involvement in the pathophysiology of Alzheimer's disease
(8, 9), induction of mRNA turnover (5), and transformation of
inhibitory postsynaptic potentials to excitatory postsynaptic
potentials (10). Although the mechanisms underlying these multiple
functions are not well understood, most of the proposed functions
appear to be mediated by Ca2+ (5).
In this paper, we describe a new homologue of CE, termed
CEB (the original form of CE (Ref. 4) is denoted
CEA for clarity). This new gene differs in nucleotide
sequence from the CEA gene, containing an insertion near
its 3' end, leading to a longer open reading frame. Our sequence
analyses indicate that CEs are members of the SCP subfamily of the
EF-hand superfamily. The recombinant CEB has been expressed
and purified from Escherichia coli and characterized using
biochemical and biophysical techniques.
Gene Cloning--
Primers were based on the DNA sequence of
CEA (accession no. U49390) (4). The sense primer (SP) had
an NdeI site and the sequence
gggaattccatatggctgcccatcaactttccgatttcc. The antisense primer (AP1) had
a BamHI site and the sequence
cgggatccgttttagggtaccaaacagatggttgccc. Polymerase chain reaction was
used, and the template was Loligo pealei optic lobe cDNA
with EcoRI adapters (provided by Dr. J. Battey, National
Institutes of Health, Bethesda, MD). Since a nucleotide addition was
always observed near the 3' end of the amplified product, the antisense
primer was changed based on the CEA sequence (4), to allow
the amplification of a product with an extended 3' sequence and the
sequence cgggatccttaaagttttagggtaccaaacagatgg. SP and the new antisense
primer (AP2) were used to amplify CEB using polymerase
chain reaction and the cDNA template described above.
CEB was cloned into a modified pGEX-2TK vector (Amersham Pharmacia Biotech, Baie d'Urfe, Quebec, Canada), which had an NdeI site (pGEX-2TK-NdeI; provided by P. Yin,
University of Toronto, Ontario, Canada), and sequenced. The deduced
amino acid sequence was aligned with ClustalX (11). Since the homology
in some regions within the sequence alignment was low, the secondary
structure assignment of the Branchiostoma lanceolatum SCP
(ASCP) (12) was used to manually adjust residues in loop regions.
Protein Expression and Purification--
E. coli BL21
cells transformed with pGEX-2TK-NdeI-CEB were
grown at 37 °C. The glutathione
S-transferase-CEB fusion protein was induced
with 1 mM isopropyl Ca2+ Overlay Blot--
CEB was run on
SDS-PAGE and transferred onto nitrocellulose (Bio-Rad, Mississauga,
Ontario, Canada). The overlay blot was performed as described
previously (14). After the blot was dried, it was subjected to
phosphorimaging (Storm 840) and analyzed by ImageQuant software
(Amersham Pharmacia Biotech).
Western Blot Hybridization--
Polyclonal anti-CEB
antibodies were prepared in rabbits with full-length CEB as
the antigen. The antibodies were affinity-purified on CNBr-activated
Sepharose 4B (Amersham Pharmacia Biotech) containing CEB.
CEB and L. pealei optic lobe (provided by Drs.
D. L. Alkon and T. J. Nelson, National Institutes of Health,
Bethesda, MD) were run on SDS-PAGE, blotted onto nitrocellulose, and
blocked with skim milk. The blot was incubated for 3 h with a
10,000-fold dilution of anti-CEB antibodies, washed, and
incubated for 3 h with a 3,000-fold dilution of horseradish
peroxidase-conjugated goat anti-rabbit IgG antibodies (Bio-Rad).
Following extensive washing, the blot was exposed using ECL
chemiluminescence (Amersham Pharmacia Biotech).
Protein Kinase C (PKC) and Casein Kinase II (CKII)
Phosphorylation--
CEB (~2 µg) was added either to
20 mM Tris-HCl (pH 8), 0.5 mM
CaCl2, 100 µg/ml phosphatidylserine (Avanti, Alabaster,
AL), 20 µg/ml diacylglycerol (Avanti), 10 mM
MgCl2, 1% bovine serum albumin, 0.1 units of PKC (Pierce),
and 125 Ci/liter [ Circular Dichroism (CD) Spectroscopy--
CD measurements were
made on an Aviv 62DS spectropolarimeter at room temperature. To study
the effect of Ca2+ on the conformation of CEB,
the protein was prepared by dialyzing against a solution containing 1 mM MES (pH 7). Samples were made with either 1 mM CaCl2 (A) or 1 mM EGTA (B). Far
UV spectra were recorded in a 0.1-cm quartz cuvette. The protein
solution was 169 µg/ml, as determined by UV absorption and described
above. The data were deconvoluted using the CD Spectra Deconvolution program (version 2.1; provided by Dr. G. Böhm,
Martin-Luther-Universität, Halle-Wittenberg, Germany). Near UV
spectra were recorded in a 1-cm quartz cuvette with a protein solution
of 2.3 mg/ml (determined by UV absorption as described above). The CD
parameters used for the phosphorylation experiment were identical to
those described above for the Ca2+ binding experiment.
Fluorescence Spectroscopy--
Steady-state fluorescence was
measured using a Photon Technology International QM-1 spectrophotometer
equipped with excitation intensity correction. Emission spectra were
collected from 305 to 400 nm ( Two-dimensional Nuclear Magnetic Resonance (NMR)
Spectroscopy--
Uniformly 15N-labeled
CEB was expressed in M9 medium (13) containing
15NH4Cl and purified as described above.
CEB was dialyzed against either a solution containing 10 mM Tris-HCl (pH 7.5), 1 mM DTT, and 10 mM CaCl2 (A) or a solution containing 10 mM Tris-HCl (pH 7.5), 1 mM DTT, and 10 mM EGTA (B). NMR sample concentration was about 1 mM 15N-labeled CEB in 5%
2H2O. The protein concentration in the
Ca2+-bound and Ca2+-free (apo) samples was
identical. The Ca2+-GTP and apo-GTP samples were prepared
by the addition of 2 mM GTP to the Ca2+ and apo
samples, respectively. The protein concentration in the Ca2+-GTP and apo-GTP samples was also identical and was
only 0.2% less than the original (Ca2+ and apo) samples,
due to the addition of GTP. NMR spectra were acquired at 37 °C on a
Varian Unity-Plus 500 spectrometer. Two-dimensional 15N-1H heteronuclear single-quantum correlation
(HSQC) spectra (16, 17) were recorded using the enhanced sensitivity
method (18), with 16 transients and 128 and 512 complex points in the
15N (F1) and 1H (F2)
dimensions, respectively.
The nucleotide sequence of the L. pealei
CEB (Fig. 1A) is
99% identical to that of CEA, with the major difference
being a single nucleotide insertion near the 3' end of the sequence.
The deduced amino acid sequence of CEB (Fig. 1B)
shows 95% identity to CEA (4). Another homologous protein
(here referred to as CET; accession no. AF078951) from the
Japanese common squid, Todarodes pacificus, has been
identified through a BLAST search. CET shows 93% amino
acid identity to CEB (Fig. 1B). Moreover, we
found that CEB shows 28% amino acid identity to ASCP (19), which is higher than the identity score (15-20%) reported among various members of the SCP subfamily (20). CEB also shows
45% amino acid identity to a neuronal SCP from Drosophila
melanogaster (dSCP2) and 18% identity to a muscle SCP from
D. melanogaster (21) (dSCP1; Fig. 1B).
CEB also shares 34% (F56D1.6, accession no. Q10131) and
28% (T09A5.1, accession no. P54961) amino acid identity to putative
proteins predicted from the Caenorhabditis elegans genome
(22) (Fig. 1B).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactoside and
after 3 h, cells were centrifuged, resuspended in 10 mM Tris-HCl (pH 7.5), 10 mM dithiothreitol
(DTT), 1 mM EDTA, 2 Complete tablets (Roche Molecular
Biochemicals, Laval, Quebec, Canada), and stored at
20 °C. Upon
thawing, cells were kept on ice, sonicated, and incubated with 1%
Triton X-100 for 30 min. The solution was centrifuged and the soluble
fraction incubated overnight at 4 °C with glutathione-Sepharose 4B
(Amersham Pharmacia Biotech) with gentle shaking. The beads containing
the bound fusion protein were washed with phosphate-buffered saline
solution (13) at 4 °C and then washed with 50 mM
Tris-HCl (pH 8), 150 mM NaCl, 0.1% 2-mercaptoethanol, 2.5 mM CaCl2 at room temperature. CEB
was cleaved from glutathione S-transferase by incubation
with 50 units of thrombin (Calbiochem, San Diego, CA) overnight at room
temperature. To the eluted protein, 1 mM
phenylmethylsulfonyl fluoride and 10 mM CaCl2
were added, checked for purity by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE), and stored at 4 °C. The
CEB N-terminal sequence was analyzed by Edman degradation
(University of British Columbia, Vancouver, British Columbia, Canada)
and its weight measured by electron-spray mass spectrometry (University
of Waterloo, Waterloo, Ontario, Canada). Both of these confirmed the
identity of CEB.
-32P]ATP (A) or to 4 mM
MES (pH 6.9), 25 mM KCl, 2 mM
MgCl2, 1 mM DTT, 0.020 milliunits CKII (Roche
Molecular Biochemicals), and 50 Ci/liter [
-32P]ATP
(B). Solutions were incubated at 37 °C for 1 h, run on
SDS-PAGE, and subjected to autoradiography, as described above. To
check the stoichiometry of PKC phosphorylation, the above reaction (A) was repeated with the substitution of [
-32P]ATP to 10 µM ATP and the molecular weight of the sample measured by
electron-spray mass spectrometry (University of Waterloo, Waterloo, Ontario, Canada). To determine the effect of PKC phosphorylation on the
conformation of CEB, the above reaction was repeated at large scale using 25 µg of CEB and 1.25 units of PKC
(Pierce). The control sample contained all the components of the
phosphorylated sample with the exception of ATP. Following incubation,
the samples and buffers were passed through a Centricon YM30 spin
column (Millipore, Bedford, MA) followed by a PD-10 column (Amersham
Pharmacia Biotech). The protein concentrations in both phosphorylated
and unphosphorylated samples were found to be identical using UV
absorption. Under denaturing conditions in 6 M
guanidine-HCl and using an extinction coefficient of
280 = 49270 M
1
cm
1, calculated by the method of Gill and von
Hippel (15), the protein concentration in these samples were found to
be 5 µM. These samples were used in the circular
dichroism and fluorescence experiments described below.
ex = 295 nm, 1 s/nm, band
pass = 2 nm for excitation and emission). For the Ca2+
binding experiments, samples were decalcified through dialysis in the
presence of EGTA followed by dialysis against decalcified water.
CEB was diluted to a final concentration of about 50 µM (as determined by UV absorption and described above)
in 100 mM KCl and 50 mM HEPES (pH 7.5). Sample
volumes were 0.5 ml, and experiments were performed at 27 °C. The
Ca2+ titration curve to calculate the Ca2+
dissociation constant (Kd) was generated in the
presence of 1.5 mM EGTA with the sequential addition of 0.2 mM CaCl2. The emission spectrum was collected
after each CaCl2 addition. Then, the fluorescence
intensities from 305 to 400 nm were integrated. The free
Ca2+ concentration was calculated based on the total
amounts of Ca2+ and EGTA present using a web-based computer
algorithm (MAXCHELATOR). The Ca2+ binding curve used
to calculate the number of Ca2+ binding sites was generated
with the sequential addition of a 10 mM CaCl2
stock (0.25-1 µl) until saturation and then with a 100 mM CaCl2 stock (0.5 µl). Sample dilution was
never more than 1.5%. The parameters used in the fluorescence
spectroscopy for the phosphorylation experiment were identical to those
described above for the Ca2+ binding experiment with the
exception of the band pass, which was set to 1 nm for both excitation
and emission.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and deduced amino acid sequence of
CEB. A, nucleotide sequence of the
full-length CEB cDNA. Nucleotide numbers are indicated,
and the stop codon is underlined. The stop codon of
CEA is indicated in bold and the major
nucleotide difference between CEB and CEA is
marked by an asterisk (*). B, the deduced amino
acid sequence of CEB is aligned with CEA (4),
calexcitin from T. pacificus (CET), the D. melanogaster neuronal dSCP2 (21), a putative C. elegans
neuronal homologue (F56D1.6) (22), a B. lanceolatum muscle
SCP isoform (ASCPIII) (19), the D. melanogaster muscle dSCP1
(21), and a putative C. elegans muscle homologue (T09A5.1)
(22). Amino acids are numbered on top, and
significant residue identities are shown in bold. The
helices found in ASCP are indicated on the top of the
alignment (H). ASCP contains only short sheet regions,
holding pairs of EF-hands together in the Ca2+ binding loop
areas (12) (these have not been indicated). A line indicates
the proposed Ca2+ binding loop in putative EF-hands.
X, Y, Z,
X, and
Z indicate potential residues involved in Ca2+
coordination, which typically are residues containing oxygen in their
side chains (Asn, Asp, Gln, Glu, Ser, and Thr) (24).
Y
indicates the sixth potential residue involved in Ca2+
coordination. G indicates the sixth residue within the loop
region, which is predominantly Gly in typical EF-hands, and
I indicates the residue that is Ile, Leu, or Val in the
canonical EF-hand (24). Potential CKII (o) and PKC
($) phosphorylation sites are shown, as determined by
PROSITE. CEB differs by one amino acid from the deduced
amino acid sequence of a gene deposited to GenBank by Nelson et
al. (accession no. AAC32271; position 11 is Lys in CEB
and Arg in AAC32271). This Lys residue has been confirmed in
CEB through limited proteolysis and N-terminal sequencing
(data not shown) and is conserved in CET and the neuronal
Drosophila SCP2. GenBank accession nos. are AF322410 for
CEB, U49390 for CEA, AAC28940 for
CET, AAB67805 for dSCP2, T30109 for F56D1.6, S13184 for
ASCPIII, AAB67804 and AE002777 (joined) for dSCP1, and T24726 for
T09A5.1.
The sequence alignment of CEB with ASCP indicates that
CEB has four putative EF-hand motifs, each motif containing
a Ca2+ binding loop and flanking helices. There is a
high sequence similarity within the EF-hand motifs, with the exception
of the fourth motif. This is due to the low sequence conservation of Ca2+ coordinating residues Asp150
(X), Leu152 (Y), Asp154
(Z), Gly156 (
Y), Thr158
(
X), and Thr161 (
Z) in
CEB (Fig. 1B). In a typical EF-hand the
X position is Asp and the Z position is Asn, Asp,
or Ser. The
Z position is always Asp or Glu, whereas the
Y position is rarely Gly, which may render the EF-hand
non-functional (23, 24). The important hydrophobic residue between
Y and
X is lacking in CEB
(Lys157; Fig. 1B). Based on this sequence
alignment and the three-dimensional structure of ASCP (12),
CEB most likely contains four helix-loop-helix structural
elements, with the fourth EF-hand incapable of binding Ca2+. This contrasts with data for CEA, which
was shown to bind only two Ca2+. This may be due to the
condition used for the experiment, in which 5 mM
MgCl2 was present. SCPs have at least one
Ca2+/Mg2+ binding site (20); hence, one of the
EF-hand sites in CEA may have been occupied with
Mg2+.
GTP-binding proteins, including ARF (25), Ras protein (26), transducin
(27), and elongation factor G (28), typically possess central structures surrounded by
helices (25-28). In all these proteins,
the GTP binding site is defined by three sequence motifs in the strict
order: GXXXXGK(S/T) (
1), DXXG (
2), and NKXD (
3) (29). These motifs are completely conserved
among all GTPases (30) and are referred to as the fingerprint of GTPase (31). In small GTPases, the
1 and
2 motifs are separated by about
40 residues and the
2 and
3 motifs by about 50-80 residues (31).
Sequence similarity of CEs to GTP-binding proteins is very low
(e.g., 11% amino acid identity to D. melanogaster ADP-ribosylation factor; Ref. 32). Despite this fact,
Nelson et al. (4) reported that CEA contains GTP
binding motifs, which are also present in CEB. However, in
both CEA and CEB, only the putative
2 GTP
binding motif is fully conserved. The Lys residue in the
3 motif is
replaced with Ile and the first Gly residue in the
1 motif is
replaced with Thr (Fig. 1B). The order of the three motifs
in the primary sequence of CEA and CEB (
2,
3, and
1) also differs from that of all other GTP-binding
proteins (
1,
2, and
3) (29). Furthermore, the
2 and
3
motifs are separated by only a single residue (Fig. 1B),
which is in disagreement with the motif spacing in small GTPases as
presented above (31). Nevertheless, Nelson et al. (4) have
shown that CEA binds GTP, although GTP binding occurred only in the absence of Mg2+. This Mg2+
inhibition of GTP binding contrasts with structural studies of various
GTPases in which Mg2+ is critical in the coordination of
the phosphate group of GTP (25-28).
Earlier studies on CEA (33) suggested that the protein
forms a homodimer, most likely due to the formation of disulfide bonds
between two CEA molecules via exposed Cys residues, in the absence of a reducing agent. Our E. coli expressed and
purified CEB showed a single band of 23 kDa on SDS-PAGE
(Fig. 2A). In addition, our
analytical ultracentrifugation experiments showed that CEB is monomeric in both the presence and the absence of Ca2+
(data not shown). This is consistent with earlier studies on SCPs,
which are generally found as monomers, with the exception of crustacean
and sandworm SCPs (20). In sandworm, Ca2+ modulates
dimerization (34), with no disulfide bond involvement (23). The
Ca2+ overlay blot of purified CEB (Fig.
2B) showed that the protein readily bound
45Ca2+. No extra band was seen, including that
corresponding to a homodimer. A polyclonal antibody that was raised
against the recombinant CEB cross-reacted with a 23-kDa
protein (Fig. 2C). The CEB antibody also
cross-reacted with a protein in the squid optic lobe (Fig. 2D). The single band found in the squid optic lobe (~22
kDa) is slightly smaller than the recombinant CEB, which is
probably due to the lack of the N-terminal addition of eight amino
acids from the glutathione S-transferase fusion protein.
When the antibody was preincubated with CEB prior to
incubation with the filter, no cross-reaction was observed on the
Western blot either with CEB or squid optic lobe tissue
(data not shown), indicating that the antibodies are specific to
CEB.
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It has been reported (4, 35) that CEA is a high affinity substrate for PKC and has a single PKC phosphorylation site (Thr61). We found that CEB was also a substrate for PKC in the presence of Ca2+ (Fig. 2E). There are two putative PKC phosphorylation sites in CEB (Thr61 and Thr188; Fig. 1B). Since CEA possesses Thr61 (4) but lacks Thr188, Thr61 may be a common phosphorylation site in CEs. Our mass spectrometry data indicate that about 80% of the protein was phosphorylated at both Thr61 and Thr188 and the remaining 20% at one of the two sites. There is no detectable amount of unphosphorylated CEB in the mass spectrum (data not shown). Whether or not the phosphorylation of these sites is functionally important remains unclear.
Previous far UV CD spectroscopy on CEA (36) has shown that
the Ca2+-free protein is composed of 28% helix and
23%
sheet and the Ca2+-bound protein is composed of
33%
helix and 18%
sheet. However, as indicated above, the
multiple sequence alignment suggested that CEB is an
-helical protein. In order to characterize the secondary structure
of CEB, we performed far UV CD spectroscopy (Fig.
3). Deconvolution of the CD data using a
neural network algorithm (37) showed that Ca2+-free
CEB contains 46%
helix and 11%
sheet, whereas
Ca2+-bound CEB contains 57%
helix and 6%
sheet. Interestingly, the [
]208 value is more
negative than the [
]222 value, atypical of the
canonical
-helical protein (38). Nevertheless, these data indicate
that CEB is highly
-helical, consistent with the sequence analysis indicating that CEB is an EF-hand
protein. Hence, we believe that the content of
sheet reported for
CEA (36) and even those obtained for CEB are
overestimated. We also suggest that the difference in
helix content
between Ca2+-free and Ca2+-bound states may
reflect changes in the orientation of preexisting
helices upon
Ca2+ binding (39). Nevertheless, the far UV CD data
indicate that CEB is folded even in the absence of
Ca2+.
|
The near UV CD spectrum of CEB (Fig.
4) differs significantly between
Ca2+-free and Ca2+-bound states, indicating
that the protein undergoes a conformational change upon
Ca2+ binding. In the region spanning 255 to 310 nm, there
are approximately nine peaks in both Ca2+-bound and
Ca2+-free CEB, due to a number of aromatic
residues (10 Phe, 7 Trp, and 7 Tyr) in CEB (Fig.
1B). All nine peaks change in magnitude upon
Ca2+ binding, suggesting that many of the aromatic residues
undergo at least minor changes in their electronic environment. The
largest change within this spectral region occurs between the
wavelengths of 275 and 300 nm, corresponding to absorbance of Trp
residues. Upon Ca2+ binding, the maximum at 280 nm
decreases in signal intensity and a minimum at 293 nm becomes apparent,
suggesting that Trp residues undergo a change in their environment.
Contribution of Phe residues is also visible in the spectra. The minima
at 261 and 267 nm are likely due to Phe residues. Minima at 263 and 268 nm are believed to be markers for the native fold (40). Furthermore, the existence of these minima suggests that at least some of the Phe
residues are buried in specific and tightly packed environments (41).
CEB contains 10 Phe residues: four in the N-terminal
half (residues 9, 18, 21, and 35) and six in the C-terminal half of the protein (residues 113, 115, 149, 165, 173, and 186; Fig.
1B). Residues 113, 165, 173, and 186 are well conserved
among neuronal SCPs (Phe165 is Lys in F56D1.6 and
Phe186 is Leu in CEA, due to the shorter C
terminus of CEA). In contrast, Phe residues 9, 18, 21, 35, 115, and 149 in CEB are well conserved across both neuronal
and muscle SCPs (residue 18 is Val and residue 21 is Met in dSCP1, and
residue 149 is Tyr in ASCPIII). Furthermore, based on the
three-dimensional structure of ASCP (12) and the multiple sequence
alignment (Fig. 1B), the ASCP residues corresponding to the
CEB Phe residues 18, 21, 35, and 115 have solvent
accessibilities of less than 3%. This suggests that these four Phe
residues are part of the hydrophobic core of CEB and
contribute to the minima around 263 and 268 nm of the near UV CD
spectra of CEB.
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CEB contains seven Trp residues: five in the N-terminal
half (residues 32, 48, 66, 86, and 90) and two in the C-terminal half of the protein (residues 106 and 169). Most of these Trp residues are
well conserved, and Trp66 is completely conserved among the
SCP proteins (Fig. 1B). As presented above, the near UV CD
data indicated that the Ca2+-induced conformational change
of CEB involves Trp residues. To confirm that the Trp
residues undergo a change in their environment, we measured the change
of fluorescence of Trp residues upon Ca2+ addition. The
intrinsic fluorescence of the Trp indole ring showed a large decrease
of fluorescence intensity (40%) upon Ca2+ addition (Fig.
5A). However, the change in
the intensity was not accompanied by a significant shift of the maximum
(max). This result is in agreement with the
aforementioned near UV CD data (Fig. 4) and NMR data (see below). It is
interesting to note that, in agreement with data presented above for
Phe residues, Trp residues that are well conserved among SCPs (residues
66, 86, 90, and 106 in CEB) have low surface
accessibilities (<7%) and hence are proposed to be part of the
hydrophobic core of the proteins.
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The change in fluorescence intensity plotted against Ca2+ concentration (Fig. 5B) shows that the midpoint of the titration occurs near 1 µM. This value is comparable to the Kd of 0.4 µM observed for CEA (4) and is in the range expected for a Ca2+-sensor protein (2). It is worth noting that the apparent Kd of Ca2+ binding to CEB is larger than the Kd observed for Ca2+ binding to SCPs, which ranges from 0.l to 0.01 µM (42). Our experiments using tryptophan fluorescence spectroscopy show that CEB binds three Ca2+ (Fig. 5C) in agreement with three conserved EF-hand Ca2+ binding loops, as described above. However, these data contrast with the previous report on CEA (4), indicating that CEA binds two Ca2+. One possibility is that the presence of 5 mM MgCl2 in the Ca2+ binding study on CEA may have contributed to this discrepancy. It has been known (42) that SCPs have Ca2+/Mg2+ binding sites.
The effect of Ca2+ binding on CEB was studied
by NMR spectroscopy (Fig. 6, A
and B). As seen in the 1H-15N HSQC
spectrum of Ca2+-free CEB (Fig. 6B),
most of the 15N-HN cross peaks are well dispersed,
indicating that the protein is folded even without Ca2+, in
agreement with the far UV CD data presented above. The addition of
Ca2+ resulted in a major spectral change, involving both
chemical shift and intensity changes for a large fraction of cross
peaks in the HSQC spectrum. In particular, the indole NH groups of
several Trp residues in CEB, resonating at >10 ppm (HN)
and >130 ppm (15N), experience a large change in chemical
shift upon the addition of Ca2+, suggesting that most, if
not all, Trp residues in CEB change their chemical
environment upon Ca2+ binding. This is in agreement with
tryptophan fluorescence spectroscopy data discussed previously (Fig.
5). Interestingly, the spectrum of Ca2+-free
CEB contains one downfield shifted peak (10.55 ppm
(HN)/117.9 ppm (15N)), whereas the spectrum of the
Ca2+-bound CEB contains three downfield shifted
peaks (10.73 (HN)/114.0 ppm (15N), 10.20 (HN)/116.5 ppm
(15N), and 10.25 (HN)/118.2 ppm (15N)). Peaks
in this region are characteristic of the residue in the sixth position
of the Ca2+ binding loop of the EF-hand domain, which is
typically Gly (1, 24). The downfield shift of these residues are likely
due to the hydrogen bond between the amide proton of the residue with the side chain of the Asp residue at the Ca2+ binding
X position of the EF-hand domain (Fig. 1B) (43).
It is worth noting that in CEB, the Gly residue of EF2 is
replaced by Glu and that of EF3 is replaced by Asn. In SCPs, the
substitution of Gly to Asn or Glu does not interfere with
Ca2+ binding (20, 42). To date, CEB and SCP
from Nereis diversicolor (44) are the only examples with
HSQC spectra showing a large downfield shift of peaks (>2 ppm in the
HN dimension) irrespective of residue type (Gly, Asn, and Glu in
CEB; Gly and Asn in SCP from N. diversicolor )
and corresponding to the sixth position of a functional
Ca2+ binding loop. These NMR data are consistent with the
tryptophan fluorescence data indicating that CEB binds
three Ca2+ ions. Furthermore, the NMR data also indicate
that two hydrogen bonds are most likely broken in the
Ca2+-free state, since only one downfield shifted peak
remains in the spectrum of Ca2+-free CEB.
|
To test the binding of Mg2+ to CEB, we recorded an HSQC spectrum of CEB in the presence of 2 mM Mg2+. The comparison of this spectrum with that of Ca2+-free CEB shows significant chemical shift changes for a number of peaks, indicating that CEB binds Mg2+. However, the changes are not as drastic as those observed when Ca2+ was added. For example, the two downfield shifted peaks that appear in the Ca2+-bound CEB spectrum (10.73 (HN)/114.0 ppm (15N) and 10.20 (HN)/116.5 ppm (15N)) are not present in the Mg2+ spectrum (data not shown). A similar experiment was performed for Ca2+-bound CEB, in which no apparent change is detected upon addition of Mg2+ (data not shown), suggesting that the Mg2+ affinity of CEB is lower than the Ca2+ affinity (Kd = 1 µM).
In contrast to the drastic Ca2+-induced change observed in
the HSQC spectrum, the addition of GTP yielded only a negligible effect
on the HSQC spectrum of CEB (Fig. 6, C and
D). Three independent GTP binding assays, nitrocellulose
filtration (45), nitrocellulose overlay (46), and GTP cross-linking
(47), did not reveal GTP binding to CEB (data not shown).
These observations and the poorly conserved GTP binding consensus
sequences in CEB, as discussed later, indicate that
CEB lacks GTP binding activity.
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DISCUSSION |
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The SCP protein subfamily of the EF-hand superfamily comprises a number of invertebrate muscle proteins, with molecular masses ranging from 20 to 22 kDa. However, there have been reports that describe SCP proteins existing in neuronal tissue (21, 48). In Helix pomatia (49) and in Aplysia californica (48), a terrestrial and a marine invertebrate, respectively, there is immunohistochemical evidence that SCPs are found in neuronal cells. Furthermore, in D. melanogaster, the gene sequences of an SCP protein from the muscle and another from the nervous tissue have been identified (with the proteins sharing only 19% identity), with no spatial overlap in expression (21). Taken together, these data suggest that a neuronal counterpart of SCP proteins may exist in invertebrates, although only a muscle isoform has been identified in the majority of organisms. It is worth mentioning that, in D. melanogaster, for which the complete genome is known (50), there is no evidence for protein isoforms, which is in agreement with a study reported previously (21).
The multiple sequence alignment also reveals that neuronal SCP proteins
share a fingerprint "YXNFM" motif in the entering EF3
helix (Fig. 1B). Based on the multiple sequence
alignment and the conservation of the fingerprint region in the EF3
motif, we propose that the putative C. elegans F56D1.6
protein is a neuronal SCP protein. The lack of the neuronal motif in
the putative C. elegans protein T09A5.1 suggests that this
protein is a muscle SCP protein. The muscle and neuronal proteins in
C. elegans are only 26% identical. These data are in
agreement with those in D. melanogaster, suggesting that,
even in the same organism, muscle and neuronal SCPs are very different.
Furthermore, there is no supporting evidence for SCP isomers (other
than the neuronal and muscle homologues mentioned above) in the known
genome of C. elegans (22), which is in agreement with the
D. melanogaster data discussed previously. Nevertheless, all
these data suggest that SCP proteins can be subdivided into two classes
based on tissue distribution and/or primary sequence. SCPs have been
thought to act as Ca2+ buffers (42), since SCPs were found
to be highly concentrated in invertebrate muscles, possessed
Ca2+/Mg2+ binding sites, and no target protein
was identified (20). To this end, it is worthwhile to point out that CE
is the first example of an SCP family member that functions as a
Ca2+ sensor. It is tempting to speculate that only neuronal
SCPs acquired Ca2+ sensor function, whereas muscle SCPs
function as Ca2+ buffers. Clearly, further investigation is
required to address this hypothesis.
It has been postulated that CEA is a membrane-binding protein (4, 33, 51, 52), because of the 3' terminal polyisoprenylation consensus domain (CAAX) and two putative myristoylation consensus sequences (4). Interestingly, the polyisoprenylation signaling sequence is absent in CEB, whereas two myristoylation consensus sequences are present in CEB. However, myristoylation can only occur at the N terminus of a mature protein (53, 54). Taken together, we believe that CEB is a cytoplasmic protein, like other SCPs (20).
PKC plays a pivotal role in activity-dependent neuronal plasticity with various pathways (55). Although PKC phosphorylates CEB (Fig. 2B), the lack of conservation of PKC phosphorylation sites even among neuronal SCPs suggests that PKC phosphorylation may not be essential for CEs. Furthermore, PKC phosphorylation failed to produce any detectable global changes in the conformation of CEB using CD or fluorescence spectroscopy (data not shown). CEB also has four putative CKII phosphorylation sites, one of which is located in the EF2 loop and conserved among all SCP homologues presented in Fig. 1B. However, CKII failed to phosphorylate either the Ca2+-bound or Ca2+-free CEB (data not shown), in agreement with the previous study of CEA (4).
CEB binds Ca2+, which leads to major
conformational changes as monitored by far and near UV CD spectroscopy,
fluorescence spectroscopy, and NMR spectroscopy. The far UV CD spectrum
of CEB (Fig. 3) is nearly identical to that reported for
CEA (36). The spectra of CEB also show a
remarkable similarity to the spectra observed with other SCPs: a high
[]222 (56-60), which is probably due to the
contribution of aromatic residues flanking the
helices (61).
Near UV CD data showed that the protein undergoes a conformational change upon Ca2+ addition, since there are spectral changes between 255 and 310 nm (Fig. 4). These data are in agreement with previous results for SCP from N. diversicolor , which showed that the spectrum between 255 and 300 nm changed dramatically with the addition of Ca2+ (34, 56). Similar changes have been observed in other EF-hand CaBPs such as S100P (62). Furthermore, near UV CD data suggest that both Ca2+-free and Ca2+-bound CEB is in a native-like conformation, due to characteristic minima around 263 and 268 nm (40).
The intrinsic tryptophan fluorescence data, in agreement with the near
UV CD data presented above, indicate that Trp residues are involved in
the Ca2+-induced conformational change in CEB.
The fluorescence spectral change comprises a reduction in intensity
with no significant shift of max. Similar spectral
change has been observed for other EF-hand CaBPs, such as scallop SCP
(57), frequenin (63), neurocalcin
and
(64), S100P (62), and
parvalbumin (65). The change in intensity of the signal combined with a
lack of shift of
max has been suggested to occur due to
a change in the electronic environment of Trp residues (65).
Interestingly, the signal intensity has decreased in three of the
aforementioned CaBPs upon Ca2+ binding. In SCP,
Ca2+ binding resulted in a signal intensity decrease of
80% (57), in frequenin a decrease of 25% (63), and in S100P a
decrease of 20% (62).
As indicated by tryptophan fluorescence spectroscopy, NMR spectroscopy suggests that Trp residues change their chemical environment upon Ca2+ binding. In contrast to tryptophan fluorescence spectroscopy, which provides information of the global changes of Trp side chains, NMR spectroscopy provides detailed information of individual Trp indole rings. However, the interpretation of the HSQC spectra is limited by the lack of resonance assignments. Nevertheless, 15N-HN cross peaks resonating at >10 ppm (HN) and >130 ppm (15N) are likely due to the indole NH groups of Trp residues. All of these Trp peaks experience a chemical shift change upon Ca2+ binding, suggesting that these Trp residues undergo changes in their chemical environment.
The Ca2+-induced NMR spectral changes of the Trp indole rings are also accompanied by major changes in the rest of the spectrum. This may indicate that Ca2+ binding induces a gross conformational change that extends to the entire molecule. Although GTP binding has been previously reported for CEA (4), based on NMR spectroscopy and binding assays we found no evidence for GTP binding to CEB. Our NMR data indicate that the Kd for GTP binding to CEB, if any, is larger than 2 mM. This is significantly larger than the Kd for specific GTP binding to other small GTPases, which ranges from 0.05 to 5 µM (66). Therefore, we conclude that CEB is not a GTP-binding protein.
In conclusion, CEB is a member of the SCP subfamily of the EF-hand superfamily, belonging to the neuronal type. CEB binds three Ca2+ ions with an apparent Kd of 1 µM. There is a conformational change induced by Ca2+, which (based on optical and NMR spectroscopies) also involves a change in the environment of many aromatic residues. Our data indicate that CEB is not a GTP-binding protein, and thus it is not a GTPase. The protein is a substrate of PKC, although this may be physiologically irrelevant.
At present, it is unclear what the biological significance of neuronal
SCP isoforms may be, especially since in Drosophila there
are no SCP isoforms in either muscle or neurons (21, 50). One
possibility may be to provide a "fine-tuning" mechanism in the
action of CE. Detailed structural and physiological studies will be
necessary to identify the mechanism(s) of action of CEB.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. B. Ames and Dr. M. B. Swindells for discussions, Dr. R. Ishima and Dr. D. Liu for help with NMR, K. Tong with laboratory help, and Dr. D. L. Alkon and Dr. T. J. Nelson with initial help with the work.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Canadian Institutes of Health Research (to A. C. and M. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF322410.
§ Supported by a Natural Sciences and Engineering Research Council postgraduate scholarship award, a Savoy Foundation for Epilepsy Studentship, and a Canadian Institutes of Health Research doctoral research award.
Supported by a Canadian Institutes of Health Research doctoral
research award.
** Supported by an Ontario Cancer Institute/Amgen postdoctoral fellowship and a National Cancer Institute of Canada research fellowship.
Recipient of Canadian Institutes of Health Research scientist
award and Howard Hughes Medical Institute international scholar award.
To whom correspondence should be addressed: Ontario Cancer Inst., Rm.
7-723, 610 University Ave., Toronto, Ontario M5G 2M9, Canada.
Tel.: 416-946-2025; Fax: 416-946-6529; E-mail: mikura@ uhnres.utoronto.ca.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M010508200
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ABBREVIATIONS |
---|
The abbreviations used are: CaBP, calcium-binding protein; CD, circular dichroism; CE, calexcitin; CKII, casein kinase II; HSQC, heteronuclear single-quantum correlation; PKC, protein kinase C; SCP, sarcoplasmic calcium-binding protein; PAGE, polyacrylamide gel electrophoresis; SP, sense primer; AP, antisense primer; ASCP, B. lanceolatum muscle SCP isoform; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Kretsinger, R. H.,
and Nockolds, C. E.
(1973)
J. Biol. Chem.
248,
3313-3326 |
2. | Ikura, M. (1996) Trends Biochem. Sci. 21, 14-17[CrossRef][Medline] [Order article via Infotrieve] |
3. | Nelson, T. J., Collin, C., and Alkon, D. L. (1990) Science 247, 1479-1483[Medline] [Order article via Infotrieve] |
4. |
Nelson, T. J.,
Cavallaro, S.,
Yi, C. L.,
McPhie, D.,
Schreurs, B. G.,
Gusev, P. A.,
Favit, A.,
Zohar, O.,
Kim, J.,
Beushausen, S.,
Ascoli, G.,
Olds, J.,
Neve, R.,
and Alkon, D. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13808-13813 |
5. | Alkon, D. L., Nelson, T. J., Zhao, W., and Cavallaro, S. (1998) Trends Neurosci. 21, 529-537[CrossRef][Medline] [Order article via Infotrieve] |
6. | Nelson, T. J., Zhao, W. Q., Yuan, S., Favit, A., Pozzo-Miller, L., and Alkon, D. L. (1999) Biochem. J. 341, 423-433[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Cavallaro, S.,
Meiri, N.,
Yi, C. L.,
Musco, S.,
Ma, W.,
Goldberg, J.,
and Alkon, D. L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9669-9673 |
8. | Kim, C. S., Han, Y. F., Etcheberrigaray, R., Nelson, T. J., Olds, J. L., Yoshioka, T., and Alkon, D. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3060-3064[Abstract] |
9. | Etcheberrigaray, E., Gibson, G. E., and Alkon, D. L. (1994) Ann. N. Y. Acad. Sci. 747, 245-255[Abstract] |
10. |
Sun, M. K.,
Nelson, T. J.,
Xu, H.,
and Alkon, D. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7023-7028 |
11. |
Thompson, J. D.,
Gibson, T. J.,
Plewniak, F.,
Jeanmougin, F.,
and Higgins, D. G.
(1997)
Nucleic Acids Res.
25,
4876-4882 |
12. | Cook, W. J., Jeffrey, L. C., Cox, J. A., and Vijay-Kumar, S. (1993) J. Mol. Biol. 229, 461-471[CrossRef][Medline] [Order article via Infotrieve] |
13. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
14. | Campbell, J. A., Biggart, J. D., and Elliott, R. J. (1991) Biochem. Soc. Trans. 19, 53S[Medline] [Order article via Infotrieve] |
15. | Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve] |
16. | Bodenhausen, G., and Ruben, D. J. (1979) Chem. Phys. Lett. 69, 185-189[CrossRef] |
17. | Zhang, O., Kay, L. E., Olivier, J. P., and Forman-Kay, J. D. (1994) J. Biomol. NMR 4, 845-858[Medline] [Order article via Infotrieve] |
18. | Kay, L. E., Keifer, P., and Saarinen, T. (1992) J. Magn. Reson. A 109, 129-133[CrossRef] |
19. | Takagi, T., and Cox, J. A. (1990) Eur. J. Biochem. 192, 387-399[Abstract] |
20. | Cox, J. A., Luan-Rilliet, Y., and Takagi, T. (1991) in Novel Calcium-binding Proteins: Fundamentals and Clinical Implications (Heizmann, C. W., ed) , pp. 447-463, Springer-Verlag, New York |
21. | Kelly, L. E., Phillips, A. M., Delbridge, M., and Stewart, R. (1997) Insect Biochem. Mol. Biol. 27, 783-792[CrossRef][Medline] [Order article via Infotrieve] |
22. |
The C. elegans Sequencing Consortium.
(1998)
Science
282,
2012-2018 |
23. | Vijay-Kumar, S., and Cook, W. J. (1992) J. Mol. Biol. 224, 413-426[Medline] [Order article via Infotrieve] |
24. | Kawasaki, H., and Kretsinger, R. H. (1994) Protein Profile 1, 343-517[Medline] [Order article via Infotrieve] |
25. | Greasley, S. E., Jhoti, H., Teahan, C., Solari, R., Fensome, A., Thomas, G. M., Cockcroft, S., and Bax, B. (1995) Nat. Struct. Biol. 2, 797-806[Medline] [Order article via Infotrieve] |
26. | de Vos, A. M., Tong, L., Milburn, M. V., Matias, P. M., Jancarik, J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E., and Kim, S. H. (1988) Science 239, 888-893[Medline] [Order article via Infotrieve] |
27. | Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 369, 621-628[CrossRef][Medline] [Order article via Infotrieve] |
28. | Czworkowski, J., Wang, J., Steitz, T. A., and Moore, P. B. (1994) EMBO J. 13, 3661-3668[Abstract] |
29. |
Kjeldgaard, M.,
Nyborg, J.,
and Clark, B. F.
(1996)
FASEB J.
10,
1347-1368 |
30. |
Cuvillier, A.,
Redon, F.,
Antoine, J.,
Chardin, P.,
DeVos, T.,
and Merlin, G.
(2000)
J. Cell Sci.
113,
2065-2074 |
31. | Dever, T. E., Glynias, M. J., and Merrick, W. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1814-1818[Abstract] |
32. | Tamkun, J. W., Kahn, R. A., Kissinger, M., Brizuela, B. J., Rulka, C., Scott, M. P., and Kennison, J. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3120-3134[Abstract] |
33. |
Nelson, T. J.,
Yoshioka, T.,
Toyoshima, S.,
Han, Y. F.,
and Alkon, D. L.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9287-9291 |
34. | Durussel, I., Luan-Rilliet, Y., Petrova, T., Takagi, T., and Cox, J. A. (1993) Biochemistry 32, 2394-2400[Medline] [Order article via Infotrieve] |
35. | Nelson, T. J., and Alkon, D. L. (1995) J. Neurochem. 65, 2350-2357[Medline] [Order article via Infotrieve] |
36. |
Ascoli, G. A.,
Luu, K. X.,
Olds, J. L.,
Nelson, T. J.,
Gusev, P. A.,
Bertucci, C.,
Bramanti, E.,
Raffaelli, A.,
Salvadori, P.,
and Alkon, D. L.
(1997)
J. Biol. Chem.
272,
24771-24779 |
37. | Greenfield, N. J. (1996) Anal. Biochem. 235, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
38. | Chen, Y. H., Yang, J. T., and Chau, K. H. (1974) Biochemistry 13, 3350-3359[Medline] [Order article via Infotrieve] |
39. | Williams, T. C., Corson, D. C., Oikawa, K., McCubbin, W. D., Kay, C. M., and Sykes, B. D. (1986) Biochemistry 25, 1835-1846[Medline] [Order article via Infotrieve] |
40. | Pain, R. (1996) in Current Protocols in Protein Science (Coligan, J. E. , Dunn, B. , Ploegh, H. L. , Speicher, D. W. , and Wingfield, P. T., eds) , pp. 761-823, John Wiley & Sons, Toronto |
41. | Strickland, E. H. (1974) CRC Crit. Rev. Biochem. 2, 113-175[Medline] [Order article via Infotrieve] |
42. | Hermann, A., and Cox, J. A. (1991) Comp. Biochem. Physiol. 111B, 337-345 |
43. | Ikura, M., Minowa, O., and Hikichi, K. (1985) Biochemistry 24, 4264-4269[Medline] [Order article via Infotrieve] |
44. | Craescu, C. T., Precheur, B., van Riel, A., Sakamoto, H., Cox, J. A., and Engelborghs, Y. (1998) J. Biomol. NMR 12, 565-566[CrossRef][Medline] [Order article via Infotrieve] |
45. | Mitchell, J., and Mayeenuddin, L. H. (1998) Biochemistry 37, 9064-9072[CrossRef][Medline] [Order article via Infotrieve] |
46. | Manser, E., Leung, T., and Lim, L. (1995) Methods Enzymol. 256, 130-139[Medline] [Order article via Infotrieve] |
47. | Tanaka, T., Saha, S. K., Tomomori, C., Ishima, R., Liu, D., Tong, K. I., Park, H., Dutta, R., Qin, L., Swindells, M. B., Yamazaki, T., Ono, A. M., Kainosho, M., Inouye, M., and Ikura, M. (1998) Nature 396, 88-92[CrossRef][Medline] [Order article via Infotrieve] |
48. | Pauls, T. L., Cox, J. A., Heizmann, C. W., and Hermann, A. (1993) Eur. J. Neurosci. 5, 549-559[Medline] [Order article via Infotrieve] |
49. | Kerschbaum, H. H., Kainz, V., and Hermann, A. (1992) Brain Res. 597, 339-342[Medline] [Order article via Infotrieve] |
50. |
Adams, M. D.,
Celniker, S. E.,
Holt, R. A.,
Evans, C. A.,
Gocayne, J. D.,
Amanatides, P. G.,
Scherer, S. E.,
Li, P. W.,
Hoskins, R. A.,
Galle, R. F.,
George, R. A.,
Lewis, S. E.,
Richards, S.,
Ashburner, M.,
Henderson, S. N.,
Sutton, G. G.,
Wortman, J. R.,
Yandell, M. D.,
Zhang, Q.,
Chen, L. X.,
Brandon, R. C.,
Rogers, Y. H.,
Blazej, R. G.,
Champe, M.,
Pfeiffer, B. D.,
Wan, K. H.,
Doyle, C.,
Baxter, E. G.,
Helt, G.,
Nelson, C. R.,
Gabor Miklos, G. L.,
Abril, J. F.,
Agbayani, A.,
An, H. J.,
Andrews-Pfannkoch, C.,
Baldwin, D.,
Ballew, R. M.,
Basu, A.,
Baxendale, J.,
Bayraktaroglu, L.,
Beasley, E. M.,
Beeson, K. Y.,
Benos, P. V.,
Berman, B. P.,
Bhandari, D.,
Bolshakov, S.,
Borkova, D.,
Botchan, M. R.,
Bouck, J.,
Brokstein, P.,
Brottier, P.,
Burtis, K. C.,
Busam, D. A.,
Butler, H.,
Cadieu, E.,
Center, A.,
Chandra, I.,
Cherry, J. M.,
Cawley, S.,
Dahlke, C.,
Davenport, L. B.,
Davies, P.,
de Pablos, B.,
Delcher, A.,
Deng, Z.,
Mays, A. D.,
Dew, I.,
Dietz, S. M.,
Dodson, K.,
Doup, L. E.,
Downes, M.,
Dugan-Rocha, S.,
Dunkov, B. C.,
Dunn, P.,
Durbin, K. J.,
Evangelista, C. C.,
Ferraz, C.,
Ferriera, S.,
Fleischmann, W.,
Fosler, C.,
Gabrielian, A. E.,
Garg, N. S.,
Gelbart, W. M.,
Glasser, K.,
Glodek, A.,
Gong, F.,
Gorrell, J. H.,
Gu, Z.,
Guan, P.,
Harris, M.,
Harris, N. L.,
Harvey, D.,
Heiman, T. J.,
Hernandez, J. R.,
Houck, J.,
Hostin, D.,
Houston, K. A.,
Howland, T. J.,
Wei, M. H.,
Ibegwam, C.,
Jalali, M.,
Kalush, F.,
Karpen, G. H.,
Ke, Z.,
Kennison, J. A.,
Ketchum, K. A.,
Kimmel, B. E.,
Kodira, C. D.,
Kraft, C.,
Kravitz, S.,
Kulp, D.,
Lai, Z.,
Lasko, P.,
Lei, Y.,
Levitsky, A. A.,
Li, J.,
Li, Z.,
Liang, Y.,
Lin, X.,
Liu, X.,
Mattei, B.,
McIntosh, T. C.,
McLeod, M. P.,
McPherson, D.,
Merkulov, G.,
Milshina, N. V.,
Mobarry, C.,
Morris, J.,
Moshrefi, A.,
Mount, S. M.,
Moy, M.,
Murphy, B.,
Murphy, L.,
Muzny, D. M.,
Nelson, D. L.,
Nelson, D. R.,
Nelson, K. A.,
Nixon, K.,
Nusskern, D. R.,
Pacleb, J. M.,
Palazzolo, M.,
Pittman, G. S.,
Pan, S.,
Pollard, J.,
Puri, V.,
Reese, M. G.,
Reinert, K.,
Remington, K.,
Saunders, R. D.,
Scheeler, F.,
Shen, H.,
Shue, B. C.,
Siden-Kiamos, I.,
Simpson, M.,
Skupski, M. P.,
Smith, T.,
Spier, E.,
Spradling, A. C.,
Stapleton, M.,
Strong, R.,
Sun, E.,
Svirskas, R.,
Tector, C.,
Turner, R.,
Venter, E.,
Wang, A. H.,
Wang, X.,
Wang, Z. Y.,
Wassarman, D. A.,
Weinstock, G. M.,
Weissenbach, J.,
Williams, S. M.,
Woodage, T.,
Worley, K. C.,
Wu, D.,
Yang, S.,
Yao, Q. A.,
Ye, J.,
Yeh, R. F.,
Zaveri, J. S.,
Zhan, M.,
Zhang, G.,
Zhao, Q.,
Zheng, L.,
Zheng, X. H.,
Zhong, F. N.,
Zhong, W.,
Zhou, X.,
Zhu, S.,
Zhu, X.,
Smith, H. O.,
Gibbs, R. A.,
Myers, E. W.,
Rubin, G. M.,
and Venter, J. C.
(2000)
Science
287,
2185-2195 |
51. | Nelson, T. J., Sanchez-Andres, J. V., Schreurs, B. G., and Alkon, D. L. (1991) J. Neurochem. 57, 2065-2069[Medline] [Order article via Infotrieve] |
52. |
Kuzirian, A. M.,
Epstein, H. T.,
Nelson, T. J.,
Rafferty, N. S.,
and Alkon, D. L.
(1998)
Biol. Bull.
195,
198-201 |
53. | Grand, R. J. (1989) Biochem. J. 258, 625-638[Medline] [Order article via Infotrieve] |
54. | Ames, J. B., Ishima, R., Tanaka, T., Gordon, J. I., Stryer, L., and Ikura, M. (1997) Nature 389, 198-202[CrossRef][Medline] [Order article via Infotrieve] |
55. | Tanaka, C., and Nishizuka, Y. (1994) Annu. Rev. Neurosci. 17, 551-567[CrossRef][Medline] [Order article via Infotrieve] |
56. | Cox, J. A., Kretsinger, R. H., and Stein, E. A. (1981) Biochim. Biophys. Acta 670, 441-444[Medline] [Order article via Infotrieve] |
57. | Collins, J. H., Johnson, J. D., and Szent-Gyorgyi, A. G. (1983) Biochemistry 22, 341-345[Medline] [Order article via Infotrieve] |
58. | Cox, J. A., Winge, D. R., and Stein, E. A. (1979) Biochimie (Paris) 61, 601-605[Medline] [Order article via Infotrieve] |
59. | Kohler, L., Cox, J. A., and Stein, E. A. (1978) Mol. Cell. Biochem. 20, 85-93[Medline] [Order article via Infotrieve] |
60. | Closset, J., and Gerday, C. (1975) Biochim. Biophys. Acta 405, 228-235[Medline] [Order article via Infotrieve] |
61. | Chakrabartty, A., Kortemme, T., Padmanabhan, S., and Baldwin, R. L. (1993) Biochemistry 32, 5560-5565[Medline] [Order article via Infotrieve] |
62. |
Gribenko, A.,
Lopez, M. M.,
Richardson, J. M., III,
and Makhatadze, G. I.
(1998)
Protein Sci.
7,
211-215 |
63. | Ames, J. B., Hendricks, K. B., Strahl, T., Huttner, I. G., Hamasaki, N., and Thorner, J. (2000) Biochemistry 39, 12149-12161[CrossRef][Medline] [Order article via Infotrieve] |
64. | Kato, M., Watanabe, Y., Iino, S., Takaoka, Y., Kobayashi, S., Haga, T., and Hidaka, H. (1998) Biochem. J. 331, 871-876[Medline] [Order article via Infotrieve] |
65. |
Pauls, T. L.,
Durussel, I.,
Cox, J. A.,
Clark, I. D.,
Szabo, A. G.,
Gagne, S. M.,
Sykes, B. D.,
and Berchtold, M. W.
(1993)
J. Biol. Chem.
268,
20897-20903 |
66. | Bobak, D. A., Bliziotes, M. M., Noda, M., Tsai, S. C., Adamik, R., and Moss, J. (1990) Biochemistry 29, 855-861[Medline] [Order article via Infotrieve] |