From the Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan
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ABSTRACT |
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Various proteins in the signal transduction
pathways as well as those of viral origin have been shown to be
myristoylated. Although the modification is often essential for the
proper functioning of the modified protein, the mechanism by which the
modification exerts its effects is still largely unknown.
Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is
involved in the synaptogenesis and neuronal plasticity, binds
calmodulin, but the protein lacks any canonical calmodulin-binding
domain. In the present report, we show that CAP-23/NAP-22 isolated from
rat brain is myristoylated and that the modification is directly
involved in its interaction with calmodulin. Myristoylated and
non-myristoylated recombinant proteins were produced in
Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain.
Phosphorylation of a single serine residue in the N-terminal domain
(Ser5) by protein kinase C abolished the binding.
Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was
also found myristoylation-dependent, suggesting the
importance of myristoylation in protein-protein interactions.
Since the finding of protein myristoylation in the catalytic
subunit of cAMP-dependent protein kinase (1), various
proteins in the signal transduction pathways as well as those of viral origin have been shown to be fatty-acylated (2). The modification is
often essential for the proper functioning of the modified protein; the
transforming activity of p60src from Rous
sarcoma virus, for example, is dependent on its myristoylation (3).
However, the mechanism by which the modification exerts its effects is
still largely unknown (2, 4).
It is generally assumed that the hydrophobic acyl groups such as
myristoyl and palmitoyl groups are often involved in the protein-membrane interactions. Due to its intermediate hydrophobicity, myristoylation has been implied in the reversible membrane association (5, 6). Studies from our own and other laboratories have established
that such a mechanism, in fact, is operative in the phosphorylation-dependent interaction of myristoylated
alanine-rich protein kinase C substrate
(MARCKS)1 with membranes (7,
8). In the case of recoverin, the binding of Ca2+
induces a drastic conformational change of the protein, and the myristoyl group hidden inside the protein will protrude from the protein and can interact with membranes (9).
However, not all the myristoylated proteins are associated with
membranes. The first protein that has been found myristoylated, cAMP-dependent protein kinase, is not a membrane protein.
In the case of calcineurin, the second protein found myristoylated, the myristoylation seems to not affect the association of the protein with
membranes, and both myristoylated and non-myristoylated proteins are
found in membrane and soluble fractions (10, 11). Interestingly, the
modification has been shown to affect the protein stability of both
proteins (10, 12). This suggests that the myristoyl moiety interacts
with the protein part in a manner analogous to that found in recoverin.
The third possible function of the protein myristoylation, namely the
involvement of the modification in protein-protein interaction, has
been the subject of various studies (10, 13-15). To our knowledge,
however, the last possibility has never been clearly demonstrated.
Brain-specific protein CAP-23/NAP-22 has been characterized first in
chicken brain as a cortical cytoskeleton-associated protein of 23 kDa
(16), and its rat homologue was later characterized as a
neuron-specific acidic protein of 22 kDa (17). It is related to another
neuron-specific acidic protein called GAP-43, a major neuronal growth
cone-associated protein (16, 17). Although the physiological function
of the protein has yet to be determined, the involvement in the
synaptogenesis and neuronal plasticity has been suggested (18). Like
the two related proteins, GAP-43 and MARCKS (19), CAP-23/NAP-22 is a
prominent substrate of protein kinase C (PKC), and the phosphorylation
by PKC is regulated by the binding to calmodulin (20). However, unlike
GAP-43 and MARCKS, which have a typical calmodulin-binding domain of
basic amphiphilic nature, CAP-23/NAP-22 lacks any canonical
calmodulin-binding domain (20).
In the present report, we show that NAP-22/CAP-23 isolated from
rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. The calmodulin-binding site of the protein was narrowed down to the myristoyl moiety together
with a nine-amino acid N-terminal basic domain. The acyl chain seems to
interact with the hydrophobic pocket of calmodulin, which is usually
occupied by the hydrophobic amino acids of target proteins.
Phosphorylation of CAP-23/NAP-22 by PKC was also found to be
myristoylation-dependent.
Preparation of Recombinant Proteins--
The complete gene of
683 base pairs including tag regions for NcoI and
XhoI restriction sites at both ends was synthesized based on
the rat cDNA sequence (17), except that Ser3 in the
deduced sequence was replaced with a Gly and that nucleotide sequence
was rewritten using the bacterial codon usage. The double-stranded DNA
was divided into 12 regions that overlap each other, and the DNA
fragments that extended from 18 to 60 nucleotides were synthesized. They were annealed, ligated, and used as template for PCR. The synthetic gene thus obtained was incorporated between the
NcoI-XhoI sites of pET14b expression vector. This
approach allowed a high expression of the protein in Escherichia
coli (86 mg/liter of culture medium). For the myristoylated
protein, a pBB131 vector containing yeast
N-myristoyltransferase cDNA was co-transformed (21).
Both proteins were purified from the heat-stable protein fractions by
successive column chromatography on Phenyl-Sepharose and Resource RPC
(Amersham Pharmacia Biotech).
Myristoylation of Peptides--
Peptides synthesized by standard
Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry
(Research Genetics, Huntsville, AL) were myristoylated by
recombinant yeast N-myristoyltransferase in vitro as described (22). The myristoylated peptides were purified over a
reversed-phase column (Vydac 218TP, 2.1 × 150 mm) using a linear
gradient of H2O-acetonitrile in the presence of 0.1% trifluoroacetic acid.
Phosphorylation by PKC--
The synthetic peptides and
recombinant proteins were phosphorylated by PKC purified from bovine
brain as has been described previously (23). The phosphorylated
peptides were purified by reversed-phase column chromatography. For the
mass spectrometric studies, the phosphorylation reaction mixtures were
directly analyzed by liquid chromatography/mass spectrometry (24).
Mass Spectrometry--
CAP-23/NAP-22 protein purified from rat
brain (24) was digested with lysyl endoprotease, and the resulting
peptide mixture was directly injected into a liquid
chromatography/electrospray mass spectrometry apparatus as described
previously (24, 25). For the sequencing of the N-terminal peptide,
fractions from the reversed-phase chromatography were combined, and
dried in a SpeedVac concentrator. The peptide was dissolved in 50%
acetonitrile containing 0.1% trifluoroacetic acid and directly infused
into the electrospray interface of a Sciex API-III mass spectrometer.
Tandem mass spectra were obtained by collision-induced dissociation of
the singly charged precursor ion (m/z 471.2) as described
(24).
Binding to Calmodulin--
Binding of the recombinant proteins
and that of peptides to calmodulin were studied using
calmodulin-agarose (Sigma). Proteins or peptides were mixed with
calmodulin-agarose in 50 mM Tris-HCl buffer (pH 6.8)
containing 1 mM CaCl2 and 0.2 M
NaCl. After a short centrifugation in a tabletop centrifuge, the
supernatants were removed to analyze the unbound fractions. After
washing twice with the same buffer, the gel-bound proteins/peptides
were eluted with 50 mM Tris-HCl buffer (pH 6.8) containing
0.2 M NaCl and 5 mM EGTA. The fractions
obtained were analyzed by SDS-gel electrophoresis.
Spectroscopic Measurements--
50 nM
dansyl-calmodulin (Sigma) was titrated with varying concentrations of
myristoylated N-terminal peptide (myr-GGKLSKKKK) in the presence of 1 mM CaCl2 as described (26). Emission spectra were recorded with the excitation wavelength set at 340 nm. Binding of
the peptides to calmodulin was monitored by recording the emission at
490 nm. Dissociation constants of calmodulin-peptide complexes were
determined by a direct fit of the data to the mass equation (26).
Circular dichroism (CD) spectroscopy of the calmodulin-peptide complex was carried out in a Jasco J-720 spectropolarimeter as described previously (26, 27).
N-terminal Myristoylation of Rat CAP-23/NAP-22--
First we have
characterized the N terminus of CAP-23/NAP-22 purified from rat brain.
The purified protein was digested with lysyl endoprotease, and the
resulting peptide mixture was subjected to the liquid
chromatography/electrospray mass spectrometric analysis. Most of the
peptides were assigned by comparing the observed masses with those
calculated from the amino acid sequence deduced from the rat cDNA
sequence (17), which established the authenticity of the protein. One
peptide eluted from the reversed-phase column near the end of the
gradient was subjected to the collision-induced dissociation (Fig.
1a). The results obtained
demonstrated clearly that the N-terminal Gly is modified with a
myristoyl group. Interestingly, the third amino acid is clearly
identified as Gly and not a Ser found in the deduced sequence (17).
Comparison of the amino acid sequences shows that the third Gly
together with other amino acids in the N-terminal region is well
conserved among CAP-23/NAP-22 proteins from various species (Fig.
1b), suggesting that the original rat cDNA sequence
contains an error at the N terminus.
Characterization of Recombinant Proteins--
To study the effects
of myristoylation on the interaction of CAP-23/NAP-22 with calmodulin,
two recombinant proteins, i.e. non-myristoylated and
myristoylated proteins, were produced in Escherichia coli as
described under "Experimental Procedures." The latter was obtained
by co-transforming the bacteria with a plasmid containing yeast
N-myristoyltransferase gene (21). The authenticity of the
two proteins thus produced was established by mass spectrometry. The
mass of the non-myristoylated protein was determined to be
21,629.2 ± 2.9 Da (theoretical mass is 21,629.1 Da for the
non-myristoylated protein with the initial Met removed), while that of
the myristoylated protein was 21,839.5 ± 2.0 Da (theoretical mass
is 21,839.5 Da). These results indicate that the two proteins start
with the second Gly residue in the reduced sequence and differ only in
the N-terminal myristoylation.
Effects of Myristoylation on the Calmodulin Interaction--
The
interaction of the recombinant proteins with calmodulin was analyzed by
binding to calmodulin-agarose beads. Myristoylated CAP-23/NAP-22 and
non-myristoylated-CAP-23/NAP-22 were mixed with calmodulin beads in the
presence of Ca2+, and the bound proteins were eluted from
the beads with EGTA. As shown in Fig.
2a, the myristoylated protein
bound to the calmodulin-agarose, and most of the bound protein was
eluted with the Ca2+-free buffer. Therefore, the binding of
CAP-23/NAP-22 to calmodulin is Ca2+-dependent,
as has been shown with the CAP-23/NAP-22 protein purified from rat
brain (20). These results also suggest that the recombinant protein is
fully functional. In contrast, the non-myristoylated protein did not
bind to the calmodulin beads to a significant extent under the same
conditions. There seems to be absolute requirement of N-terminal
myristoylation in the CAP-23/NAP-22-calmodulin interaction.
Binding of N-terminal Peptides to Calmodulin--
To
elucidate the mechanisms underlying the
myristoylation-dependent calmodulin binding, peptides were
synthesized based on the N-terminal sequence and myristoylated using a
purified recombinant N-myristoyltransferase in vitro. Their
binding to calmodulin was assessed using calmodulin-agarose as above.
Surprisingly, not only the longest peptide synthesized containing the
N-terminal 18 residues (GGKLSKKKKGYNVNDEKA) but also the shortest
peptide of nine amino acids (GGKLSKKKK) showed a similar
Ca2+-dependent binding to calmodulin (Fig.
2b). Corresponding non-myristoylated peptides did not show
any significant binding to calmodulin. The myristoylated peptide can
fully mimic the behavior of the intact protein, suggesting that the
myristoylated N-terminal domain is solely responsible for the binding
of CAP-23/NAP-22 to calmodulin.
A more quantitative analysis was carried out by measuring the
fluorescence change of dansyl-calmodulin upon binding of the target
peptide (26). While the addition of the myristoylated N-terminal
peptide caused a drastic increase in the intensity and a shift of the
peak maximum of the emission spectra, little, if any, change was
observed with the addition of the non-myristoylated peptide (Fig.
2c). This is in good agreement with the direct binding results studied with calmodulin-agarose, and suggests that the binding
of the myristoylated peptide is associated with a conformational change
of calmodulin. Using the fluorescence change, the dissociation constant
of the short peptide, myr-GGKLSKKKK, was determined from the titration
data to be 3.0 nM, which is similar to that observed with
the intact myristoylated protein (20). These values suggest that the
binding of the myristoylated peptide to calmodulin is among the highest
of calmodulin-binding proteins (28). Although calmodulin has been known
to bind to phospholipids through hydrophobic interactions, the
affinities toward the membrane lipids are very low (29). Furthermore,
the addition of myristic acid to calmodulin did not affect the
calmodulin fluorescence (data not shown). These results suggest that
the binding of CAP-23/NAP-22 to calmodulin involves specific
interactions rather than simple hydrophobic interactions between
calmodulin and the myristoyl moiety.
Identification of the Calmodulin-binding Domain of NAP-22--
To
elucidate the amino acid residues involved in the binding, the
myristoylated peptide (myr-GGKLSKKKK) was partially digested with lysyl
endoprotease and a mixture of myristoylated peptides of varying length
was produced. Mass spectrometric analysis showed that the mixture
contained myr-GGKLSKKKK, myr-GGKLSKK, myr-GGKLSK, and myr-GGK. The
mixture was incubated with calmodulin-agarose, and the unbound and
bound fractions were analyzed by mass spectrometry (Fig.
3). The longest peptide was almost
completely bound to the agarose, and no significant amounts of the
peptide was detected in the unbound fraction. In contrast, the shortest
peptide, myr-GGK, did not show any significant binding under the
conditions employed. The two other peptides showed intermediate binding
characteristics (50% and 30% binding for myr-GGKLSKK and myr-GGKLSK,
respectively, calculated from the peak intensities). The affinity of
the peptides in the decreasing order is myr-GGKLSKKKK > myr-GGKLSKK > myr-GGKLSK > myr-GGK. Clearly, the presence
of four consecutive Lys residues is important for the binding. Since
the shortest peptide, myr-GGK, did not bind to calmodulin to
significant extent, the binding of the myristoylated N-terminal
peptides is effected not only by simple hydrophobic interaction between
the myristoyl group and the hydrophobic part of calmodulin, but also
involves specific interactions. In this context, it is of interest to
note that the replacement of the second Gly with a Ser, which was found in the original deduced sequence, resulted in the reduced binding affinity by 1 order of magnitude (data not shown). These results again
support our view that the peptide moiety plays an important role in the
interaction, and that specific structural requirements exist to achieve
a high affinity binding of nanomolar range.
N-terminal Peptide Assumes Non-helical Structure in Calmodulin
Complex--
To understand the structural basis of CAP-23/NAP-22
binding to calmodulin, CD spectra of the myristoylated N-terminal
domain peptide and those of calmodulin complex were measured. It has been shown that peptides derived from calmodulin-binding domain of
target proteins assume random structures in solution, but form Effects of Phosphorylation on Calmodulin
Binding--
Phosphorylation of the intact protein by PKC has been
shown to abolish its interaction with calmodulin (20). We have examined whether the N-terminal peptide (myr-GGKLSKKKK) can be
phosphorylated by PKC, and if it can, whether the phosphorylation
affects the binding of the myristoylated N-terminal peptide with
calmodulin. Both myristoylated and non-myristoylated N-terminal
peptides were stoichiometrically phosphorylated by PKC as shown
by mass spectrometry (Fig.
5a). Since the peptides
contained only one phosphorylatable residue, we concluded that
Ser5 is the phosphorylation site by PKC. Phosphorylation by
PKC abolished the binding of the peptide to calmodulin completely, as
is the case with the intact myristoylated protein (Fig. 5b).
From these results, we conclude that the myristoylated N-terminal basic
domain is the sole calmodulin-binding and PKC phosphorylation domain of
CAP-23/NAP-22, and that the interaction with calmodulin is regulated by
the phosphorylation of a single Ser in the domain.
Effects of Myristoylation on PKC-dependent
Phosphorylation--
Since the N-terminal myristoylated domain is
phosphorylated by PKC, it is of interest to examine whether the
phosphorylation is affected by myristoylation. Both myristoylated and
non-myristoylated CAP-23/NAP-22 were incubated with purified PKC, and
the resulting mixtures were analyzed by mass spectrometry. As shown in
Fig. 6, both proteins were
stoichiometrically phosphorylated, but there was a clear difference in
the time course. The myristoylated protein was almost completely
phosphorylated after 10-min incubation, while only 60% of the
non-myristoylated protein was phosphorylated at that time. Therefore,
the myristoylation seems to facilitate the interaction of CAP-23/NAP-22
with PKC.
In the present report, we could narrow down the calmodulin-binding
domain of CAP-23/NAP-22 to the myristoyl moiety together with the
nine-amino acid N-terminal domain (myristoyl-GGKLSKKKK). Even a shorter
peptide (myristoyl-GGKLSK) binds to calmodulin albeit weakly.
Addition of each basic Lys increases the affinity to calmodulin
incrementally. Since a myristoylated peptide consisting of the
myristoyl moiety and the first 18 N-terminal amino acids showed an
affinity similar to that of the nine-amino acid peptide (data not
shown), the first 9 amino acids should suffice for calmodulin binding.
As is the case for the typical calmodulin-binding motif (31), the
interaction between the basic residues of the binding protein and the
acidic residues of calmodulin seems to play an important role in the
binding. However, a more striking result is the fact that the binding
of the intact CAP-23/NAP-22 protein as well as that of the N-terminal
peptide is dependent on the presence of the myristoyl moiety.
A closer look at the N-terminal sequence immediately shows that the
domain contains one hydrophobic residue (Leu) in addition to the five
basic amino acids (Lys). This is actually reminiscent of the canonical
calmodulin-binding motif, a basic amphiphilic motif, in which basic
hydrophilic residues and hydrophobic ones appears alternatingly (28,
32). In the calmodulin-binding domain of CAP-23/NAP-22, a
hydrophobic acyl group is followed by a basic Lys residue, which is
followed by a hydrophobic Leu residue. If one assumes that the
acyl group can substitute large hydrophobic amino acids such as Trp and
Leu found in the canonical binding motif, the overall structural
characteristics are very similar. A canonical motif, however, would
span a minimal length of 12 amino acids between the two critical
hydrophobic residues (31). However, when the CAP-23/NAP-22 peptide
assumes an elongated structure, the distance between the myristoyl
moiety and Leu4 is comparable to that between the two
critical hydrophobic amino acids found in the target proteins (Fig.
7). A CD spectrum of the myristoylated
peptide-calmodulin complex, in fact, suggests that the peptide assumes
an elongated structure rather than typical
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Myristoylation and N-terminal sequence of rat
CAP-23/NAP-22. a, tandem mass spectrum of the
N-terminal peptide of CAP-23/NAP-22 purified from rat brain. Purified
protein was digested with lysyl endoprotease, and the N-terminal
peptide was isolated by reversed-phase column chromatography. The
peptide isolated was subjected to the collision-induced dissociation as
described under "Experimental Procedures."
bn and yn ions denote the
N-terminal and C-terminal fragments, respectively. b,
comparison of the N-terminal domain of CAP-23/NAP-22 from various
species. Ser2 that is mistakenly read in the rat sequence
is underlined. The initial Met that is cut off before
myristoylation is omitted. Sequences were from Ref. 17 (a),
accession number P80723 (b), accession number 1930063 (c), and Ref. 16 (d).
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Fig. 2.
Effects of myristoylation on the interaction
with calmodulin. a, myristoylated- and
non-myristoylated recombinant CAP-23/NAP-22 proteins to
calmodulin-agarose (27). b, binding of myristoylated and
non-myristoylated N-terminal peptide, myr-GGKLSKKKK. c,
fluorescence emission spectra of dansyl-calmodulin in the presence and
absence of the N-terminal peptides (28).
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Fig. 3.
Effect of varying length of the N-terminal
peptide on calmodulin binding. A myristoylated peptide,
myr-GGKLSKKKK, was partially digested with lysyl endoprotease. After
stopping the reaction by boiling, the obtained peptide mixture was
either directly injected to the liquid chromatography/mass spectrometry
apparatus (total), or treated with calmodulin-agarose. The fraction not
bound to the agarose (unbound), or that bound and eluted from
calmodulin-agarose with EGTA (bound) was analyzed by mass spectrometry
as described under "Experimental Procedures." Original electrospray
mass spectra were deconvoluted.
-helical structures in calmodulin complex. Since the overall secondary structures of calmodulin, and hence the
-helical content, do not change appreciably, changes in CD spectra associated with complex formation are mainly attributed to the change in the target proteins (30). CD spectrum of the equimolar complex of the N-terminal peptide and calmodulin was compared with the mathematical sum of those
of the individual components. As shown in Fig.
4, there was practically no change upon
the complex formation. The peptide alone in solution showed a CD
spectrum with a single negative peak below 200 nm, which is
characteristic for a random coil (data not shown). These results
suggest that the conformation of the myristoylated peptide do not
change upon calmodulin binding, and the peptide assumes a non-helical
conformation in the complex.
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Fig. 4.
CD spectra of myristoylated peptide in
complex with calmodulin. Figure shows CD spectrum of CAP-23/NAP-22
N-terminal peptide and calmodulin complex after mixing ( ). CD
spectra obtained with calmodulin and myristoylated peptide were
mathematically added (
).
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Fig. 5.
Effects of PKC phosphorylation on the
calmodulin-binding of the N-terminal peptide. Myristoylated
peptide, myr-GGKLSKKKK, was phosphorylated by PKC, and purified over a
reversed-phase column chromatography. The original peptide
(a) and phosphorylated peptide (b) were analyzed
by electrospray mass spectrometry as described under "Experimental
Procedures." The binding of the phosphorylated peptide was assessed
using calmodulin-agarose.
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Fig. 6.
Effect of myristoylation on the
PKC-dependent phosphorylation of CAP-23/NAP-22
protein. Both myristoylated and non-myristoylated recombinant
CAP-23/NAP-22 proteins were phosphorylated with purified PKC for
indicated times, and the phosphorylation was analyzed by mass
spectrometry (29).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix. Therefore, it is
reasonable to assume that the acyl chain interacts with the hydrophobic
pocket in the C-terminal lobe of calmodulin, while the Leu4
interacts with that in the N-terminal lobe in a manner analogous to the
other canonical calmodulin-binding motif (31). In fact, the hydrophobic
caves offered by calmodulin for the hydrophobic interactions have very
flexible structures and can fit a large variety of target proteins.
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Fig. 7.
Comparison between the canonical
calmodulin-binding peptide and the myristoylated CAP-23/NAP-22
peptide. Space-filling model of the M13 peptide derived from
skeletal muscle myosin light chain kinase s in an -helical
conformation (top). Hydrophobic amino acids that play
important roles in the calmodulin interaction are shown in
red. Myristoylated N-terminal peptide of CAP-23/NAP-22
(myr-GGKLSKKKK) is in an elongated structure (bottom).
Myristoyl moiety and Leu4 are shown in red. The
models were rendered on an IRIS Indigo2 workstation (SGI) using the
Insight II (Molecular Simulations) and SYBYL/Base softwares (Tripos,
Inc.).
CAP-23/NAP-22 is not the only example of calmodulin target protein that assumes a non-helical structure in the calmodulin complex. We have recently shown that the calmodulin-binding domain of MARCKS binds to calmodulin in a non-helical elongated structure despite the classical basic amphiphilic character (27). We have also pointed out the presence of a novel class of calmodulin-binding proteins that assume non-helical structures in calmodulin complex. The calmodulin-binding domains of these proteins are characterized by the dominance of basic amino acids contrary to the canonical motif in which hydrophobic amino acids are dominant (27). It is, of course, not easy to estimate the hydrophobicity of the CAP-23/NAP-22 calmodulin-binding domain, which clearly constitutes a third class of calmodulin-binding proteins. However, the fact that the myristoyl group has only weak or intermediate hydrophobicity compared with large hydrophobic amino acids (5, 6), and the fact that the basic amino acids are important in the CAP-23/NAP-22-calmodulin interaction, may suggest a close relationship of CAP-23/NAP-22 to the non-classical class of the target proteins. The elucidation of the three-dimensional structures should yield a more insight into the structural consideration of these proteins.
Protein myristoylation has been implicated in the regulation of various signal transduction proteins (4, 5). It is obvious that the modification is essential for the membrane targeting of these proteins (4, 7). Interestingly, the presence of a myristoyl group is not enough for the stable membrane anchoring of these proteins. A basic amino acid cluster is often found near the acyl group (33), and the two domains work cooperatively in the reversible membrane binding of these proteins (34). Either protein phosphorylation or calmodulin binding regulates the reversible membrane binding. Since CAP-23/NAP-22 seems to be anchored to membrane through its N-terminal domain (35), and since the same domain binds calmodulin and PKC, the membrane association of CAP-23/NAP-22 may be regulated in a similar manner.
In contrast to the role in the membrane binding, the role of protein
myristoylation in the protein-protein interaction is still largely
unknown. However, the three-dimensional structure of recoverin revealed
the presence of direct interaction of the acyl group with its own
protein moiety (9, 36). The effects on protein stability found in
cAMP-dependent protein kinase and calcineurin also imply
some kind of interaction between the myristoyl group and protein
parts. Furthermore, we have already shown that the N-terminal
myristoylation modulates the MARCKS-calmodulin interaction (37), and
the involvement of the modification in the protein-protein interactions
has also been speculated in G proteins (38). In the present report, we
have also shown that the myristoylation affects the phosphorylation of
peptides by PKC. Therefore, protein myristoylation may be also involved
in the protein-protein interaction between PKC and its substrate proteins. In fact, potentiation of PKC inhibitor peptides by N-terminal myristoylation has been reported (39, 40), and even the presence of a
myristyl binding site in PKC has been speculated (41). Altogether,
these results raise the possibility that the protein myristoylation
plays direct roles in the protein-protein interaction of various proteins.
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ACKNOWLEDGEMENT |
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We thank Dr. J. I. Gordon for providing N-myristoyltransferase expression vector.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Fujita Health University, by Grants-in-aid for Scientific Research on Priority Areas 07279105, 08249104, and 09235231, and by Grant-in-aid for Scientific Research C 09680775 from the Ministry of Education, Science, Sports and Culture, Japan.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.
To whom all correspondence should be addressed: Institute for
Comprehensive Medical Science, Fujita Health University, Kutsukake, Aichi 470-1192, Japan. Tel.: 81-562-93-9381; Fax: 81-562-93-8832; E-mail: htanigut{at}fujita-hu.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: MARCKS, myristoylated alanine-rich protein kinase C substrate; PKC, protein kinase C; CD, circular dichroism; PCR, polymerase chain reaction; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
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