(Received for publication, November 4, 1994; and in revised form, January 16, 1995)
From the
Two neuronal protein kinase C substrates, RC3/neurogranin and
GAP-43/neuromodulin, preferentially bind to calmodulin (CaM) when
Ca is absent. We examine RC3
CaM and
GAP-43
CaM interactions by circular dichroism spectroscopy using
purified, recombinant RC3 and GAP-43, sequence variants of RC3
displaying qualitative and quantitative differences in CaM binding
affinities, and overlapping peptides that cumulatively span the entire
amino acid sequence of RC3. We conclude that CaM stabilizes a basic,
amphiphilic
-helix within RC3 and GAP-43 under physiological salt
concentrations only when Ca
is absent. This provides
structural confirmation for two binding modes and suggests that CaM
regulates the biological activities of RC3 and GAP-43 through an
allosteric, Ca
-sensitive mechanism that can be
uncoupled by protein kinase C-mediated phosphorylation. More generally,
our observations imply an alternative allosteric regulatory role for
the Ca
-free form of CaM.
Calmodulin (CaM) ()activates numerous proteins in
response to Ca
fluxes. Its N- and C-terminal globular
domains, which are connected by a flexible,
-helical
``tether,'' each contain a pair of helix-loop-helix-binding
sites with slightly different Ca
affinities (for
reviews, see Forsén et al., 1986; Means
and Conn, 1987; Cohen and Klee, 1988; Means, 1988; Strynadka and James,
1989; McPhalan et al., 1991; Kretsinger, 1992; Weinstein and
Mehler, 1994). Occupation of at least the high affinity pair by
Ca
causes the flexible tether to bend, bringing the
globular domains closer together to form a more compact structure. At
the same time, more subtle rearrangements within each globular domain
cause increased exposure of hydrophobic side chains (LaPort et
al., 1980; Tanaka and Hidaka, 1980). Collectively, these
conformational changes enable CaM to bind and activate many classes of
proteins, including protein kinases, inositol triphosphate kinase,
nicotinamide adenine dinucleotide kinase, calcineurin, calcium pumps,
cyclic nucleotide phosphodiesterase, cyclases, nitric oxide synthase,
and cytoskeletal proteins (Kincaid and Vaughan, 1986; Tallant and
Cheung, 1986; Ryu et al., 1987; Cohen and Klee, 1988; Means,
1988; Bredt and Snyder, 1990; Klee, 1991; Carafoli et al.,
1992). Transient activations of some or all of these proteins are
essential for the development, growth, and environmental adaptation of
virtually all cell types, as well as for plasticity within the
mammalian central nervous system.
Two neuronal proteins are unusual
because they bind CaM with a greater affinity in the absence of
Ca (Andreasen et al., 1983; Baudier et
al., 1991). One is GAP-43, also known as neuromodulin or b-50,
which is associated with prenatal axonal growth and can be induced in
adults by axonal injury (for reviews see Skene, 1989; Liu and Storm,
1990; Benowitz and Perrone-Bizzozero, 1991; Coggins and Zweirs, 1991;
Gispen et al., 1991; Strittmatter et al., 1992).
GAP-43 associates with the cytoplasmic face of axonal growth-cone
membranes and has been implicated in presynaptic events that contribute
to synaptic development and such neuroplastic phenomena as neurite
extension (Doster et al., 1991; Fitzgerald et al.,
1991; Meiri et al., 1991; Sommervaille et al., 1991;
Lin et al., 1992), modulation of neurotransmitter release (De
Graan et al., 1991; Dekker et al., 1991) and long
term potentiation (Lovinger et al., 1985; Gianotti et
al., 1992). The second protein, known as RC3, neurogranin, or
BICKS, is a neuron-specific, postnatal-onset protein which accumulates
in forebrain dendritic spines, loosely associated with postsynaptic
structures (Watson et al., 1990, 1992; Represa et
al., 1990; Baudier et al., 1991; Coggins et al.,
1991). It is probably involved in neonatal synaptogenesis and has been
implicated in the post-synaptic second messenger cascade of long term
potentiation (Klann et al., 1992; Chen et al.,
1993a).
Recently one other protein, the neuron-specific Drosophila protein igloo (Neel and Young, 1994) has
been reported to interact preferentially with CaM when Ca is absent. It contains three regions that are homologous with the
overlapping PKC recognition and CaM-binding domains of RC3 and GAP-43.
Other proteins have been described that bind to CaM regardless of
ambient Ca
, but these interactions are either
Ca
-independent or stronger when Ca
is present (Sharma and Wang, 1986; Ladant, 1988; Dasgupta et
al., 1989). We will use the term ``Ca
sensitive'' to refer to interactions that require the
absence of Ca
.
The CaM-binding regions of RC3 and
GAP-43 share primary sequence similarities with those of proteins that
interact with CaM only when Ca concentrations are
high: many basic amino acids, no acidic amino acids, and an abundance
of hydrophobic residues with a periodicity suggestive of an amphiphilic
-helix (Anderson and Malencik, 1986; Blumenthal and Krebs, 1988;
O'Neil and Degrado, 1990). In the presence of
Ca
, CaM binding stabilizes a basic, amphiphilic
-helix within target proteins that can be detected by circular
dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies (Cox et al., 1985; DeGrado et al., 1985; McDowell et
al., 1985; Klevit et al., 1985; Seeholzer et al. 1986; MacLachlan et al., 1990; Ikura et al.,
1992a, 1992b; Precheur et al., 1991, 1992; Vorherr et
al., 1990; Munier et al., 1993; Zhang et al.,
1993; Zhang and Vogel, 1994a, 1994b). The overlapping CaM-binding and
PKC recognition domains of both RC3 and GAP-43 are predicted to form an
-helix when analyzed by the method of Garnier et
al.(1978) or with the Gascuel and Golmard (1988) basic statistical
method. A pair of strongly hydrophobic amino acids separated by 8 or 12
residues (Ile
or Phe
, and Ile
in
RC3, Ile
or Phe
, and Leu
in
GAP-43) may correspond to residues in
Ca
/CaM-activated proteins that interact with one or
both of the pliant hydrophobic patches exposed by CaM in response to
Ca
(Ikura et al., 1992a; Meador et
al., 1993).
RC3 and GAP-43 belong to a family of proteins that
bind CaM or CaM-like molecules and share an IQ motif with a consensus
sequence of IQxxxRGxxxR (Mercer et al.,
1991; Cheney and Mooseker, 1992). The isoleucine in this consensus
sequence corresponds to Ile in RC3 and Ile
in
GAP-43. This motif is found in p68 RNA helicase (Buelt et al.,
1994) and in heavy chains of many myosins in regions that interact with
light chains (light chains are structurally and functionally similar to
CaM). Xie et al.(1994) solved the structure of scallop myosin
and observed that the two light chains stabilize a long
-helix
within the heavy chain containing its IQ motifs.
We recently
characterized the RC3CaM interaction by fluorescence emission
spectroscopy and proposed that RC3 acted as a capacitor for CaM
availability within dendritic spines (Gerendasy et al., 1994).
That study, which utilized purified recombinant RC3 and sequence
variants containing amino acid substitutions within their CaM-binding
domains (Gerendasy et al., 1995), suggested that there were
two CaM-affinity forms of RC3, but did not address directly the
structural basis of these binding modes. To evaluate the hypothesis
that CaM binding stabilizes a helix within RC3 and GAP-43, we
investigated the binding interactions by CD. We conclude that CaM
mediates the Ca
-sensitive stabilization of basic,
amphiphilic
-helices within RC3 (residues 26-47) and GAP-43
(residues 31-52) under physiological salt concentrations. When
Ca
is present, RC3 and GAP-43 associate with CaM with
little or no detectable stabilization of structure. Our findings
provide structural support for the notion of two CaM affinity forms of
RC3 and, by extension, GAP-43, and further suggest a mode by which
their activities might be regulated. More generally, they suggest that
CaM induces a helical conformation within certain target proteins only
when Ca
is low or absent, implying that the
Ca
-free form of CaM may also be an active regulatory
molecule.
Figure 1:
Changes in dichroism caused by
RC3CaM, GAP-43
CaM, and RC3
CaM
interactions and TFE. Mean residue ellipticities
([
]MRE), calculated as described under
``Experimental Procedures,'' were plotted as a function of
wavelength (nm). A and B, 3.5 µM RC3,
1.25 µM GAP-43, or 3.5 µM peptide
RC3
(indicated) was placed in one compartment of
a tandem cuvette and an equimolar concentration of CaM was placed in
the other. CD spectra were collected before (solid lines) or
after (dotted lines) mixing the components of the two
compartments, in the presence of 20 µM EGTA (A)
or 5 mM Ca
(B). Amplitude
differences between the RC3
spectra displayed in panels A and B are due to experimental variability
between individual mixing experiments. C, the CD spectrum of
10 µM RC3, 5 µM GAP-43, or 71 µM RC3
was obtained in the presence of 0, 20,
40, 50, 60, and 80% TFE. Spectra derived in the absence of TFE or in
the presence of 80% TFE are indicated. Those generated in the presence
of 20, 40, 50, or 60% TFE fall between these two spectra in descending
order when viewed from 205 to 240 nm.
Figure 2:
Effect of Ca, KCl, and
intermolecular interactions on the conformations of RC3, GAP-43, and
CaM. A and B, RC3 or CaM (indicated), in the presence
of EGTA, was placed in one compartment of a tandem cuvette and buffer
containing 5 mM Ca
(A) or 150
mM KCl (B) was placed in the other. CD spectra were
collected before (solid lines) or after (dotted
lines) mixing the components of each compartment. C and D, mixing experiments were performed between CaM and RC3 (C) or GAP-43 (D), as described in Fig. 1and
under ``Experimental Procedures,'' except that increasing
concentrations of KCl (0, 50, 100, 150, 200, 250, 300, and 400
mM) were included in both compartments of the tandem cuvette.
CD spectra were collected before (open circles) or after (closed triangles) mixing the components of each compartment.
Pre- and post-mix spectra generated in the absence of KCl are
indicated. With increasing concentration of KCl, the pre-mix spectra
tend to descend and the post-mix spectra tend to ascend when viewed
between 205 and 240 nm. E, summary of mixing experiments
displayed in panels C and D. Mean residue
ellipticities, measured at 222 nm ([
]MRE
)
are plotted against concentration of KCl. Data derived from
interactions between CaM and GAP-43 (ovals) or RC3 (rectangles) are indicated. Light stippling is used
within symbols that represent pre-mix data points while dark
stippling is used in symbols representing post-mix data points. F, fluorescence emission spectroscopy of 9AC in the presence
of Ca
/CaM, Ca
/CaM+RC3,
EGTA/CaM, or EGTA/CaM+RC3 was performed as described under
``Experimental Procedures.''
The CD spectra
generated individually by RC3, GAP-43, and RC3 showed marginal negative ellipticities at 222 nm (Fig. 1C; top spectrum in each panel)
suggesting a general lack of structure for each species. The spectra
generated by RC3 was not altered appreciably by KCl (Fig. 2B), the presence of reducing agents, changes in
pH, or by changes in protein concentration (data not shown). Mixing RC3 (Fig. 2A) or GAP-43 (data not shown) with
Ca
to a final concentration of 2 mM also had
no appreciable effect. Examination of CaM by itself revealed
conspicuous minima at approximately 222 and 205 nm, characteristic of
an
-helical conformation, which increased significantly at 2
mM Ca
(Fig. 2A).
When
RC3, GAP-43, or RC3 was placed in one chamber
and equimolar CaM in the other, each spectrum corresponded to the sum
of the spectra of the individual components (Fig. 1,A and B). Because RC3 and RC3
are
small and relatively unstructured compared to CaM, their respective
pre-mix spectra were dominated by that of CaM. On the other hand,
GAP-43 which is larger and proportionately less structured makes a
greater contribution to the pre-mix spectra. After mixing, no
differences in spectra were observed in the presence of Ca
(Fig. 1B); however, markedly different spectra
were seen when Ca
was absent (Fig. 1A). The differences involved significant
enhancement of negative ellipticities between wavelengths 205-240
nm, with minima at approximately 205 and 222 nm, characteristic of an
increase in
-helical content. These enhanced spectra resembled
those obtained when Ca
was present (Fig. 1B), which are dominated by the contribution from
the Ca
-CaM interaction (Fig. 2A).
Calcium had little effect on the spectra generated by RC3 or GAP-43
alone, raising the disturbing possibility that the RC3, GAP-43, and
RC3 preparations contained contaminating
Ca
or the intriguing possibility that these proteins
caused conformational changes in CaM by mimicking Ca
.
The first possibility is unlikely. During purification, RC3 and GAP-43
were precipitated with trichloroacetic acid, resuspended in buffers
containing EGTA, and never again exposed to Ca
.
Furthermore, RC3
and other synthetic peptides
were screened for ionic impurities by ion mass spectroscopy and found
to be uncontaminated. Thus, the proteins themselves appear to be
responsible for changes in conformation.
The second possibility,
that RC3 or GAP-43 induce conformational changes in CaM analogous to
those caused by Ca, is inconsistent with the
following experimental data: calcium induces both gross and fine
conformational changes within CaM. At the gross level these changes can
be imitated by salt, a phenomenon described previously by Hennessey et al.(1987). As salt-induced CaM
-helicity reaches
saturation, the ability of Ca
to promote additional
-helicity disappears and, at physiological salt concentrations,
Ca
fails to elicit a response that can be detected by
CD spectroscopy. Whereas increasing ionic strength had no effect on RC3
-helical content, it caused a detectable increase in that of CaM (Fig. 2B). To evaluate the effect of KCl on the
structural consequences of complex formation, we performed a series of
mixing experiments with RC3 or GAP-43 in one chamber of a tandem
cuvette and CaM in the other and varied the concentrations of KCl
equally in both chambers. Due to the strong contribution of CaM to the
pre-mix spectra, the minima at 205 and 222 prior to mixing increased
with salt concentration until saturation was reached (Fig. 2, C-E). The change in ellipticity that was caused by mixing CaM
and GAP-43 diminished with increasing salt concentrations until a KCl
concentration of 200 mM, at which point no difference could be
discerned between pre- and post-mix spectra (Fig. 2E).
On the other hand, the interaction between RC3 and CaM continued to
produce a change in ellipticity, even when 400 mM KCl was
present (Fig. 2E). At this concentration of salt, CaM
does not exhibit increased negative ellipticity in response to addition
of Ca
. When similar mixing experiments were performed
in the presence of Ca
, little if any difference could
be discerned between pre- and post-mix spectra (data not shown).
Assuming KCl and Ca
have similar effects, the
observed increase must have been caused either by the stabilization of
an
-helical conformation within RC3 or structural changes within
CaM that are fundamentally different than those caused by
Ca
.
KCl does not cause CaM to activate
Ca/CaM-dependent enzymes, so more subtle
conformational changes must also be mediated by Ca
and these have been detected using various fluorescent probes
(Forsén et al., 1986). For example, at
physiological salt concentrations, Ca
-induced
conformational changes within CaM cause exposure of hydrophobic clefts
within each globular domain that results in an increased quantum yield
of the fluorescence probe (9AC) (LaPort et al., 1980). Since
the exposure of a pliant hydrophobic face appears to be a prerequisite
for the activation of Ca
/CaM-dependent enzymes, we
tested RC3 for ability to induce such structural transformations by
monitoring RC3-induced changes in emitted fluorescence from a mixture
of CaM and 9AC (Fig. 2F). When Ca
was
added to the mixture, the quantum yield increased as expected.
Inclusion of RC3 in either the presence or absence of Ca
quenched the signals slightly, perhaps due to interactions
between RC3 and CaM or to environmental effects. Nevertheless, RC3 did
not increase fluorescence emission in the absence of
Ca
. Thus, interactions between CaM and RC3, GAP-43,
or RC3
lead to an increased
-helicity within
one or both molecules only when Ca
is absent, and the
effect is different than that which occurs when Ca
binds to CaM.
To map the region(s)
of RC3 responsible for inducing global increases in negative
ellipticity, we compared nine peptides, with amino acid sequences that,
cumulatively, span the entire sequence of RC3, for their capacities to
increase -helicity when mixed with CaM (Fig. 3,
5A, and Table 1). In the absence of Ca
(Fig. 3A), RC3
showed an
85% increase in negative ellipticity when mixed with CaM, while
peptides RC3
, RC3
, and
RC3
showed changes on the order of 40%. No
appreciable changes were observed with the other five peptides when
mixed with CaM in the absence of Ca
, and none of the
nine provoked responses greater than 10% when Ca
was
present (Fig. 3B).
Figure 3:
The CaM-binding domain mapped with
peptides. Panels A-C summarize the peptide data in Table 2. A and B, percent change in mean
residue ellipticity observed at 222 nm
([]MRE
) when the indicated peptides were
mixed with CaM in the absence (A) or presence (B) of
Ca
. C, [
]MRE
of each peptide in the presence of 0, 20, 40, 60, and 80% TFE
(from left to right). In the case of RC3
and
RC3
, spectra were also obtained in the presence
of 50% TFE.
CD spectra of each of the
peptides were also obtained individually and in the presence of
increasing concentrations of TFE (Fig. 3C, 5A,
and Table 1). Prior to the addition of TFE, peptides
RC3, RC3
, and
RC3
exhibited mean residue ellipticities
(MRE[
]
expressed in deg cm
dmol
) of approximately -1.0
10
, while peptides derived from more C-terminal sequences
displayed mean residue ellipticities of approximately -0.4
10
to 0.4
10
. The CD spectra of
these peptides suggest a random coiled conformation (data not shown).
When increasing amounts of TFE were added, RC3
,
RC3
, RC3
,
RC3
, and RC3
responded with
increasing negative ellipticities, indicating a propensity for an
-helical conformation. The remaining four peptides showed no
appreciable sensitivity to TFE. In the presence of 80% TFE, peptide
RC3
exhibited the greatest helical content
followed closely by that of RC3
, while those of
RC3
, RC3
, and
RC3
were relatively small. Nevertheless, all five
peptides showed large proportional responses in that 80% TFE induced
negative ellipticities manyfold higher than that displayed in a
completely aqueous environment. Thus, with the exception of
RC3
, which lacks crucial CaM-binding residues,
the helical propensity and degree of fluidity displayed by individual
peptides correlated closely with their ability to evoke a change in
-helicity when interacting with CaM in the absence of
Ca
. Control experiments performed in the presence of
dithiothreitol ruled out the possibility that spectral changes resulted
from the formation of cystine dimers (data not shown).
Figure 4:
Interaction between CaM and RC3, RC3
sequence variants, or GAP-43. Percent change in mean residue
ellipticity observed at 222 nm ([]MRE
)
when the indicated proteins were mixed with CaM in the absence (left panel) or presence (right panel) of
Ca
. Percentages are calculated from data in Table 1
If CaM
stabilizes an -helix within the CaM-binding domains of RC3 and
GAP-43, mutations that enhance Ca
-sensitive binding,
such as the substitution of Ala for Ser
in RC3 might be
expected to do so by augmenting helical tendencies within the binding
domain. To test this hypothesis we examined the negative ellipticities
of RC3 and sequence variants F37W, S36A, S36K, and S36D with and
without TFE. (Table 2). We also compared the ellipticities of
peptide RC3
and one corresponding to the same
region of S36A (S36A
) (Table 1). Although
S36A and S36A
appeared to exhibit greater
negative ellipticities than RC3 and RC3
,
respectively, the differences fell within the standard errors of the
amino acid content analysis used to determine protein and peptide
concentrations.
Spectroscopy of RC3 or GAP-43
alone indicates that their individual conformations are not
significantly affected by Ca or salt, despite their
high degree of structural fluidity and strong predilection for the
-helical conformation, as demonstrated by TFE studies. Mapping
experiments with peptides demonstrated, with one exception, a
correlation between those regions exhibiting structurally fluid
-helical tendencies and those that elicit
Ca
-sensitive increases in negative ellipticity when
interacting with CaM. The exception was RC3
,
which exhibited a high degree of fluidity and strong
-helical
tendencies but failed to interact with CaM, almost certainly because of
the absence of residues 43 through 47 (RKKIK). These residues are
conserved in GAP-43 and required for its binding to CaM (Alexander et al., 1988; Baudier et al., 1991; Chapman et
al., 1991). This highly basic stretch of amino acids is probably
required for initial anchoring of RC3 or GAP-43 to CaM as discussed
below.
The data summarized above constitutes strong circumstantial
evidence that an -helix is stabilized within the CaM-binding
domains of RC3 and GAP-43 upon binding to CaM when Ca
is absent and is consistent with the helical structure observed
in the heavy chain of scallop myosin (Xie et al., 1994),
another protein that contains an IQ motif. The conclusion is further
supported by its ability to explain the dissimilar CaM binding
affinities of sequence variants F37W and S36A in spite of the close
proximity and similarly hydrophobic nature of their respective sequence
substitutions (the former exhibits a greater
Ca
-independent affinity while the latter displays an
increased Ca
-sensitive affinity). One would expect
that a Ser-to-Ala substitution would be helix stabilizing while a
Phe-to-Trp substitution would not (Marqusee, et al., 1989;
Padmanabhan et al., 1990). Increasing the hydrophobic bulk of
residue 37 could enhance Ca
-independent binding while
replacing Ser
with a helix-promoting residue would
increase Ca
-sensitive binding. Unfortunately, the
methods used here lack the necessary precision to demonstrate increased
-helicity on the part of S36A. Interactions between F37W or GAP-43
and CaM generated only a small or undetectable increase in
-helicity, respectively, when Ca
was present,
even though a significant portion of each would be expected to form a
complex with CaM, independent of Ca
levels (both have
micromolar dissociation constants in Ca
). In low
ionic environments such as those used here, RC3, S36K, and S36A also
interact, although to lesser extents, in Ca
and
induce small changes in ellipticity when they associate with CaM (S36D
has the lowest affinity and elicits the smallest change in
ellipticity).
Figure 6:
Three views of the putative -helix
formed by RC3 residues 25-47. The helix on the left is oriented
with Ser
to the front. From this point of view one can
discern a hydrophilic (blue), positively charged (+) face
that includes Ser
. The middle helix has been rotated
clockwise 120° and the helix on the left another 120°. The
right-most helix is oriented with Phe
to the front. A
predominantly hydrophilic face (yellow) which includes
Phe
is visible in the helix on the
right.
Figure 5:
Graphic representation of peptide data. A-C, nine overlapping peptides that collectively span the
entire 78-residue sequence of RC3 are represented by rectangles. The data are extracted from Table 2. A, []MRE
of each peptide prior
to the addition of TFE. B, [
]MRE
of each peptide in the presence of 80% TFE. C, percent
change in [
]MRE
that occurred when each
peptide was mixed with CaM in the presence of EGTA. The PKC recognition
and minimal CaM-binding domains (Apel et al., 1990; Alexander et al., 1988; Baudier et al., 1991; Chapman et
al., 1991; Houbre et al., 1991) are indicated at the top
of each panel. D, primary structure of RC3 and homologous
region of GAP-43. The overlapping PKC recognition and CaM-binding
domains are indicated over the primary structure of RC3. The minimal
CaM-binding domain as determined by Alexander et al.(1998) and
Chapman et al.(1991) is indicated by a solid line while additional residues deemed to be important for
Ca
-sensitive interactions with CaM based on the data
presented here are indicated by a dotted line. E,
hypothetical secondary structure of the that region. The helical
propensity exhibited by each peptide, inferred from its behavior in
TFE, and each peptide's individual ability to elicit a
Ca
-sensitive increase in helicity when mixed with
CaM, suggests that sequences N-terminal to Ser
, which
occupy a predominantly random coiled conformation (left), are
able to transiently sample a helical conformation (center).
Direct binding of CaM, in the absence of Ca
, to
residues C-terminal to Ser
are hypothesized to stabilize a
helix on the C-terminal side. This propagates and is further stabilized
by the formation of a helix N-terminal to Ser
(right).
Negative
ellipticities exhibited by RC3,
RC3
but not RC3
when
exposed to TFE or CaM suggest that amino acids N-terminal to Ser
(ANAAAAKIQAS) contribute substantially to the formation of an
-helix. There are three chemically non-conservative amino acid
differences between RC3 and GAP-43 in this region (Fig. 5D), which could account for their different
affinities for CaM and the sensitivity of those affinities to salt. RC3
and GAP-43 differ from other known CaM-binding proteins in that their
CaM-binding domains only assume a stable amphiphilic,
-helical
conformation when interacting with CaM in the absence of
Ca
. Sequences N-terminal to Ser
may be
responsible for this unconventional behavior.
Interactions between
RC3 or GAP-43 and CaM, PKC, and possibly membrane phospholipids may be
mediated through positively charged and hydrophobic faces of the
-helix (Fig. 6). This would be consistent with the
hypothesis that ACTH antagonizes GAP-43
CaM and GAP-43
PKC
interactions through its presumed ability to form a competing
amphiphilic
-helix (Zweirs and Coggins, 1991). It is also
consistent with the observation that RC3 and de-myristalted GAP-43
associate with liposomes composed of acidic phospholipids (Houbre et al., 1991). In this model, Lys
,
His
, Arg
, Lys
, Lys
,
and Lys
constitute one positively charged surface that
winds around one side of the helix, unbroken except for
Ser
, the target of PKC. Phosphorylation of Ser
or its substitution with aspartate would place a negative charge
in the center of this positively charged face, possibly disrupting
interactions with CaM. Residue 36 appears to be pivotal for the
propagation of an
-helix, thus explaining how its phosphorylation
or substitution with aspartate abrogates both low and high affinity
binding. Chen et al.(1993) have demonstrated that Lys
is important for PKC specificity, implying that the positively
charged face is important for recognition by PKC. Ser
also
lies immediately adjacent to another continuous surface composed of
hydrophobic residues Ala
-Ala
,
Ile
, Phe
, Met
, Ala
,
and Ile
. The amino acid differences in GAP-43 do not alter
the nature of these surfaces. Mutagenesis and fluorescence binding
studies with peptides and recombinant proteins indicate that at least
one of the residues within the hydrophobic face, Phe
in
RC3 or Phe
in GAP-43, interacts with CaM and with PKC
(Alexander et al., 1988; Chapman et al., 1991, Chen et al., 1993; Gerendasy, et al., 1994).