From the Departments of Biochemistry and
¶ Morphology, Faculdade de Medicina de Ribeirão Preto,
Universidade de São Paulo, Ribeirão Preto,
São Paulo 14049-900, Brazil
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ABSTRACT |
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Myosin-V, an unconventional myosin, has two
notable structural features: (i) a regulatory neck domain having six IQ
motifs that bind calmodulin and light chains, and (ii) a structurally distinct tail domain likely responsible for its specific intracellular interactions. Myosin-V copurifies with synaptic vesicles via its tail
domain, which also is a substrate for calmodulin-dependent protein kinase II. We demonstrate here that myosin-V
coimmunoprecipitates with CaM-kinase II from a Triton X-100-solubilized
fraction of isolated nerve terminals. The purified proteins also
coimmunoprecipitate from dilute solutions and bind in overlay
experiments on Western blots. The binding region on myosin-V was mapped
to its proximal and medial tail domains. Autophosphorylated CaM-kinase
II binds to the tail domain of myosin-V with an apparent
Kd of 7.7 nM. Surprisingly, myosin-V
activates CaM-kinase II activity in a
Ca2+-dependent manner, without the need for
additional CaM. The apparent activation constants for the
autophosphorylation of CaM-kinase II were 10 and 26 nM,
respectively, for myosin-V versus CaM. The maximum
incorporation of 32P into CaM-kinase II activated by
myosin-V was twice that for CaM, suggesting that myosin-V binding to
CaM-kinase II entails alterations in kinetic and/or phosphorylation
site parameters. These data suggest that myosin-V, a
calmodulin-carrying myosin, binds to and delivers CaM to CaM-kinase II,
a calmodulin-dependent enzyme.
The primary intracellular receptor for Ca2+ in
neuronal cells is calmodulin, which mediates the calcium signal by
reversible, Ca2+-regulated binding to many target enzymes,
which include the calmodulin-dependent protein kinases
(reviewed in Ref. 1). Recently, unconventional myosins that carry
calmodulins as light chains have been identified in nervous tissue and
implicated in neuronal processes such as phototransduction in retina
(2, 3), growth cone dynamics (4, 5), and synaptic function (6, 7). One
of these, a class V myosin from brain
(BM-V),1 is an oligomeric
molecule composed of two identical heavy chains of 212 kDa, and 12-14
light chains in the 10-20-kDa range including multiple calmodulins
(reviewed in Refs. 8-11). The native molecule thus has two
mechanochemical head domains each bearing a well conserved ATP-binding
site, an actin-binding site, and sequence homology with other known myosins.
There are two notable structural features of this myosin. 1) A
regulatory neck domain is composed of six tandem IQ motifs, which are consensus sequences for light chain binding (12). Calmodulin
is the major light chain (13) and copurifies with BM-V at a
stoichiometry of about 4 mol/mol of heavy chain (14). The
actin-activated, MgATPase activity of BM-V is tightly and cooperatively
regulated by Ca2+, presumably via its binding to calmodulin
(15). Recently, light chains, homologous to the essential light chains
of conventional myosins, have also been shown associated with
BM-V.2 2) A tail domain,
having intercalated coiled-coil and globular segments, is structurally
distinct from that of other classes of myosins and thought to be
responsible for the specific intracellular interactions and/or
locations of this myosin. Very little is known about its structure and
properties, although we have shown it to be phosphorylated by the
multifunctional, calmodulin-dependent protein kinase II
(CaMKII) (17). No functional significance for this fact has yet been assigned.
CaMKII is an abundant protein in brain with a broad substrate
specificity. It associates with the actin cytoskeleton (18), as well as
with synaptic vesicles (19) and postsynaptic densities (20), and is
involved in neuronal processes such as neurotransmitter synthesis and
release, ion channel regulation, and long term potentiation (reviewed
in Refs. 1 and 21). Its kinase activity is regulated by
Ca2+/calmodulin and autophosphorylation in a complex manner
(see Ref. 1). In a cellular milieu, how CaMKII is regulated, what it phosphorylates, and when may be determined in part by the subcellular colocalization of the participating components, including calmodulin as
well as specific substrates. A general role for the
calmodulin-carrying, unconventional myosins in the subcellular
localization of calmodulin has been suggested (22).
In this paper we present biochemical evidence, which suggests that BM-V
can bind to and deliver calmodulin to a calmodulin-requiring enzyme,
CaMKII. We show that CaMKII coimmunoprecipitates with BM-V from a
Triton X-100-soluble extract of synaptosomes. Furthermore, the purified
proteins bind to each other in both immunoprecipitation and overlay
experiments. We use the overlay technique to determine the binding
affinity and to map the binding site(s) on the BM-V molecule. Finally,
we show that both the autophosphorylating activity of CaMKII and
substrate phosphorylation of BM-V are activated in this complex.
Materials--
[ Antibodies--
The following monoclonal antibodies were
obtained commercially: anti- Purification of Proteins--
Myosin-V was purified from chick
brain essentially as described by Cheney (24). Approximately 8 calmodulins copurify per BM-V with this procedure (14), which includes
several precipitation steps and two cromatography columns, indicating
their tight association with BM-V. Final fractions containing pure BM-V
were pooled, dialyzed against 20 mM imidazole-HCl, pH 7.4 (containing 75 mM KCl, 2.5 mM
MgCl2, 0.1 mM EGTA, 1 mM DTT), and
stored up to 2 weeks at 4 °C or with 30% sucrose at
Calmodulin was purified from bovine brain, either fresh or frozen, by
the method of Gopalakrishna and Anderson (25), followed by purification
on HPLC to remove low molecular weight contaminants, as follows: 0.5 ml
of calmodulin (0.2 mM in 26 mM HEPES, pH 7.2, containing 0.5 mM EGTA) from the Gopalakrishna and Anderson
preparation was loaded onto a MonoQ HR 5/5 column (Amersham Pharmacia
Biotech) and eluted with a gradient of NaCl (0-487 mM in 8 min; 487-503 mM in 4 min; 503-750 in 3 min). Calmodulin
elutes at about 495 mM NaCl and is separated from a closely
pre-eluting contaminant by judicial fraction collection and
reapplication to the column. The final preparation of calmodulin is
highly purified as judged by Coomassie Blue and silver staining on low
molecular weight gels.
CaMKII was purified from rat forebrains by calmodulin affinity
chromatography as described by Goldenring et al. (26) with modifications. Rat forebrains in lots of 5 were rapidly removed and
homogenized with an Omni high speed tissue grinder in 10 ml of ice-cold
50 mM imidazole-HCl, pH 6.9, containing 10 mM
EDTA, 10 mM EGTA, 2 mM DTT, 1 mM
Pefabloc, 2.0 µg/ml aprotinin, and 1 mM benzamidine. All
further steps were done at 4 °C unless otherwise stated. The
homogenate was centrifuged at 40,000 × g for 20 min and the supernatant recentrifuged at 100,000 × g for
1 h. The resulting supernatant was applied at a flow rate of 30 ml/h to a phosphocellulose column (60-ml bed volume) equilibrated in 50 mM imidazole-HCl, pH 6.9, containing 1 mM EDTA
and 2.0 mM DTT. The column was washed with 5 volumes of
equilibration buffer and eluted with the same buffer adjusted to pH 8. Eluting fractions were assayed for
Ca2+/calmodulin-dependent autophosphorylation
of 50-60-kDa polypeptides characteristic of CaMKII, or otherwise
assayed by probing dot blots of the column fractions with a monoclonal
antibody against the Cloning and Expression of Fusion Proteins--
Molecular cloning
techniques used were essentially as described by Sambrook et
al. (27). The subcloning of the head, neck, and complete tail
domains of chicken BM-V in fusion with maltose-binding protein (MBP; 42 kDa) in the pIH902 vector was described by Espreafico et al.
(13). The medial tail and globular tail domains were subcloned in
fusion with glutathione S-transferase (GST; 27.5 kDa) in the
pGEX vector (Amersham Pharmacia Biotech). The sequence corresponding to
the proximal tail domain (nt2811-3440) was amplified by polymerase
chain reaction using primers containing restriction sites for
EcoRI at their ends. The amplified fragment was digested with this enzyme and inserted into the EcoRI site of the
pIH902 vector. The predicted molecular mass of this fusion protein is 65.2 kDa (aa 911-1122). The medial tail domain was derived from the
32a clone in pBS vector (13), digested with XmnI. The
fragment of 962 base pairs (nucleotides 3425-4387) was purified and
inserted into the SmaI site of the pGEX3 vector. The
predicted molecular mass of the expressed fusion protein is 62.5 kDa
(aa 1117-1435). The sequence encoding the globular tail was also
derived from the 32a clone, digested with HpaI and
EcoRV, resulting in a 1.2-kilobase pair fragment that was
inserted into the SmaI site of the pGEX3 vector. The
predicted molecular mass of this fusion protein is 70.4 kDa (aa
1440-1830). All of the fusion proteins mentioned above were expressed
in Escherichia coli XL1Blue strain. The whole tail of BM-V
was also subcloned and expressed using the pET vector, which resulted
in its fusion at the N terminus with a 16-amino acid segment derived
from the T7 vector. This construct was the same as that cloned in
fusion with MBP in the pIH902 vector and was produced by cutting the 8c
clone in pBS vector (13) with EcoRI and ligating this
fragment (nucleotides 2778-6599) into the EcoRI site of
pET5a. This whole tail fragment encodes 102.4 kDa of BM-V (aa
899-1830) and was expressed using the pLysS strain of E. coli. The proteins mentioned above were expressed after induction
by IPTG and were identified in SDS-PAGE by comparison of non-induced
and induced bacterial lysate samples and by immunoblotting when appropriate.
Immunoprecipitation--
Antibodies were purified by affinity
column chromatography as described by Espreafico et al.
(13). The flow-through fraction from the affinity column did not
recognize BM-V in Western blots and was therefore used as the
"preadsorbed" serum control. Pansorbin cells were washed in 10 volumes of TBS/Tween (40 mM Tris buffer, pH 8.0, containing
150 mM NaCl and 0.5% Tween 20) by several cycles of
centrifugation and resuspension. Aliquots of affinity-purified antibody
or preadsorbed serum were incubated with the washed cells for 2 h
at room temperature in the proportion of 2 mg of protein/ml of
Pansorbin cells. The cells were then washed three times with 10 volumes
of TBS/Tween. Protein samples in microcentrifuge tubes were incubated
for 2 h at room temperature with 50 µl of the antibody-laden Pansorbin cells under gentle agitation. The cells were collected by a
60 s spin in a microcentrifuge (this first supernatant, referred to as "s" in the figures, was saved and analyzed
together with the final immunoprecipitate, "p") and
washed by three centrifugation/resuspension cycles in TBS/Tween.
Finally, the wash supernatant was carefully removed and the Pansorbin
pellet dissolved in SDS-PAGE sample buffer, boiled for 2 min,
centrifuged for 2 min in a microcentrifuge, and this supernatant
(referred to as "p" in the figures) analyzed by SDS-PAGE
and immunoblotting using appropriate antibodies.
"Overlay" Technique--
Western blots of protein samples on
reinforced nitrocellulose membranes (0.22 µm) were blocked by
incubation with 5% nonfat dried milk in TBS/Tween for at least 2 h. The protein fractions to be used as probes were incubated with the
Western blot under gentle agitation for 2-2.5 h. The filters were then
washed with TBS/Tween with three quick changes, followed by four washes
of 5 min each. Binding of the probing protein to polypeptides on the
Western blot was detected in two ways: 1) by incubating the filter with
probe-specific antibodies for 1 h and revelation with alkaline
phosphatase-conjugated secondary antibodies, or 2) if the protein probe
was radioactive, by autoradiography of the dried blot.
Assay for Phosphorylation--
Reaction mixtures containing 25 mM imidazole-HCl, pH 7.4, 1 mM EGTA, 4 mM MgCl2, and 1 mM DTT were
prepared in microcentrifuge tubes. CaMKII, BM-V, calmodulin, 100 µM free Ca2+, and 50 µM
trifluoperazine (TFP) were added as indicated in the figure legends. To
obtain 100 µM free Ca2+, CaCl2
was added based on the calculation of the buffering capacity of 1 mM EGTA by Fabiato (28). The reaction, at 35 °C, was
initiated and stopped in two ways depending on the experiment: 1)
reactions containing all other ingredients were initiated by addition
of ~2 µCi of [ Other Methods--
Synaptosomes were prepared from rat cerebral
cortex and purified over a Percoll gradient as described by Nagy and
Delgado-Escueta (29). SDS-PAGE was performed using 6-15% linear
gradient minigels (30). Immunoblotting was done as described by Towbin
et al. (31), using alkaline phosphatase-conjugated secondary
antibodies developed by the nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate. The free
Ca2+ concentration indicated in the experiments was based
on the calculation of 1 mM EGTA buffer capacity as
described by Fabiato (28). Concentrations of BM-V and calmodulin were
based on the following extinction coefficients: BM-V, 1.04 at 280 nm
for 1.0 mg/ml solutions (14); calmodulin (in 1 mM EGTA),
2.0 at 276 nm for 1% solution (32). Otherwise, protein concentration
was determined by the method of Bradford (33) using bovine serum
albumin, fraction V, as a standard. The molecular weight of native BM-V
was taken as 640,000 (14) and CaMKII holoenzyme as 600,000 (26).
BM-V Coimmunoprecipitates with CaMKII and Syntaxin from a Triton
X-100-solubilized Extract of Synaptosomes--
Synaptosomes from rat
cerebral cortex were isolated on a Percoll gradient and examined by
Western blotting for the presence of myosins and other proteins (Fig.
1). Myosins II and V were most
concentrated in the 20% Percoll interphase, which corresponded to the
highly enriched synaptosome fraction as verified by electron microscopy
(Ref. 28; data not shown). The exclusion of neurofilament protein
(NF200) and enrichment of synaptophysin, an integral membrane protein
of synaptic vesicles, served as biochemical markers in this fraction
and corroborated the integrity of the preparation. CaMKII was
distributed throughout the gradient.
In a previous study (6), we showed that BM-V was partially solubilized
by Triton X-100 extraction of lysed synaptosomes in the presence of
millimolar concentrations of ATP. Since Triton extraction disrupts
membranes, while many protein-protein interactions are maintained, we
attempted to identify potential protein-ligands of BM-V in the
Triton-solubilized fraction of synaptosomes by immunoprecipitation with
antibodies against the BM-V tail domain. By immunodetection on Western
blots, we were able to show coimmunoprecipitation of CaMKII and
syntaxin, a synaptic plasma-membrane protein, with BM-V under these
conditions (Fig. 2). The portion of the
total CaMKII and syntaxin in the soluble fraction that cosedimented with BM-V was small; however, the fact that synaptophysin, also present
in equivalent amounts in the synaptosome lysate, did not cosediment
with these proteins argues against nonspecific aggregation. Additionally, none of these proteins were cosedimented with Pansorbin cells coated with pre-adsorbed antiserum. CaMKII and syntaxin participate in large, variably assembled protein and cytoskeleton complexes, formed during the cycle of events of synaptic transmission (reviewed in Ref. 34). Thus, these results suggest that BM-V may also
be an eventual component of these supramolecular structures.
Purified CaMKII Coimmunoprecipitates with BM-V or Its Bacterially
Expressed Tail Domain--
To assay for direct binding between BM-V
and CaMKII, we performed in vitro coimmunoprecipitation
experiments from a dilute mixture of the two purified proteins using
the BM-V tail antibodies (Fig.
3A). Coimmunoprecipitation of
CaMKII and BM-V was observed in the absence of ATP (reaction 2, no
phosphorylation condition) as well as under conditions where
autophosphorylation was promoted (reaction 3). Both non-phosphorylated
(seen in lanes 2p and 3p) and
phosphorylated forms of
To permit direct demonstration of the incorporation of 32P
into the phosphorylated species, reaction mixtures of BM-V and CaMKII, diluted 2- and 8-fold in relation to the CaMKII concentration of Fig.
3A, were incubated for 1 min at 37 °C upon addition of 10 µM [ CaMKII Binds to the BM-V Heavy Chain and Its Bacterially Expressed
Tail Domain; BM-V Binds to Both Autophosphorylated CaMKII Binds to the BM-V Whole Tail Domain with
High Affinity, and the Binding Site Is Centered around the Proximal
Tail Region--
In order to estimate the affinity of the CaMKII
binding to BM-V, we developed a method of ligand detection on the
immobilized tail domain with the overlay technique using
autophosphorylated [32P]CaMKII as the ligand probe. As
shown in Fig. 6A, analysis of the overlaid blot by phosphor image scanning demonstrated that [32P]CaMKII was bound to the immobilized tail at
incubation concentrations in the nanomolar range. The data from a
typical experiment, plotted in Fig. 6B, showed an apparently
hyperbolic increase in [32P]CaMKII binding with increased
concentration. The reciprocal plot of the data (inset) gave
an apparent Kd of 7.7 nM. Four
equivalent experiments gave Kd values that ranged from 1.1 to 7.7 nM. In order to compare the binding of
non-phosphorylated versus autophosphorylated CaMKII,
equivalent overlay experiments were done except that they were probed
with anti-
Based on these experiments, we attempted to map the binding site on the
BM-V molecule by examining the binding of [32P]CaMKII to
recombinant proteins corresponding to several regions of BM-V (Fig.
7A). Strong binding was
observed on the whole tail domain (lane 8) and
the proximal tail segment (lane 5). Weaker, but
clearly detectable binding was observed for the medial tail region
(lane 6). No binding was detected over the head
(lane 4) and globular tail (lane
7) segments, or over the GST or MBP proteins alone or
proteins in the non-induced bacterial extract. We also have detected
very weak binding to the expressed neck domain in separate experiments
(data not shown), but due to difficulties in the bacterial expression
of this domain, we have not obtained clearly comparative results. These
composite data are illustrated on a linear map of BM-V (Fig.
7B) and suggest that the binding site for CaMKII is centered
around the proximal tail region with some participation from adjacent
regions.
BM-V Activates the Protein Kinase Activity of CaMKII without
Requirement for Exogenous Calmodulin--
Previous work from our
laboratory showing that BM-V is a substrate for CaMKII (17) was
confirmed here in Fig. 4 using purified proteins. In order to better
characterize this activity in terms of Ca2+ and calmodulin
requirements, we performed phosphorylation reactions with the purified
proteins under several conditions (Fig.
8). The surprising result was that a
mixture of CaMKII and BM-V showed Ca2+-dependent autophosphorylation of the
kinase subunits and substrate phosphorylation of BM-V without
requirement for exogenous calmodulin. Since this activity was abolished
by micromolar concentrations of the calmodulin antagonist,
trifluoperazine, these data suggest that BM-V, which has calmodulin
light chains bound to its neck domain, was contributing one or more of
its calmodulins to the regulatory domain of CaMKII. The
immunoprecipitation of BM-V from aliquots used in kinase activation
experiments resulted in the diminution of kinase activity (data not
shown), thus indicating that the presence of native BM-V was necessary
for the full activation effect on CaMKII.
In order to quantitate this activation, the incorporation of
32P into CaMKII induced by BM-V was determined. The time
course of the incorporation was not linear under the conditions studied (Fig. 9) and, in fact, suggested the
occurrence of a burst of autophosphorylating activity within 15 s
of initiation of the reaction. We therefore used the shortest reaction
time attainable in practice (15 s) to determine the effect of
increasing BM-V concentration on 32P incorporation. The
activation of CaMKII by BM-V, together with that by exogenous
calmodulin on the same preparation of CaMKII and under the same
reaction conditions, is illustrated in Fig. 10A. Reciprocal plots of
these data, shown in Fig. 10B, gave values of 10 and 26 nM for the apparent activation constants of BM-V and
calmodulin, respectively, which is consistent with an exchange of about
2-3 calmodulins from BM-V to CaMKII under the conditions of the
experiment. Notably, the maximum incorporation of 32P into
CaMKII activated by BM-V was about twice that by exogenous calmodulin.
Although the protocol used here does not permit us to distinguish
between the initial steady-state rate and a possible initial burst
phase (Fig. 9), the data suggest that BM-V not only supplies
calmodulin, but additionally the binding of BM-V to CaMKII entails
alterations in kinetic and/or autophosphorylation site parameters.
An important target of Ca2+/calmodulin in neuronal
cells is CaMKII, whose kinase activity is stringently regulated by the
Ca2+-dependent binding of calmodulin. CaMKII is
a multifunctional enzyme with a wide amplitude of potential cellular
substrates (reviewed in Refs. 1, 21, and 35). Thus, its subcellular localization and specific associations within its immediate molecular vicinity are likely to be important factors in its cellular function and control. In the pre-synaptic region, for example, CaMKII is bound
to synapsin I and synaptic vesicles where it regulates, via
phosphorylation, the interactions between these components and the
actin cytoskeleton (36, 37), an important regulatory process of
neurotransmitter release (reviewed in Ref. 38). CaMKII is also
associated with postsynaptic densities, submembranous actin-cytoskeleton structures believed to be involved in receptor regulation and synaptic plasticity (reviewed in Refs. 39 and 40).
Autophosphorylation of CaMKII affects its reversible association with
postsynaptic densities (41) and recent studies identified the
polypeptides, p190 and p140, as major CaMKII-binding proteins (42).
Calmodulin would not be expected a priori to have limited
access to target proteins, being small, highly soluble, and present in
relatively high concentrations in brain (43). However, in the cell
calmodulin is indeed discretely localized to several subcellular
compartments, for example, postsynaptic densities (44), centrosomes and
the mitotic apparatus (45), the contractile vacuole of
Dictyostelium (46), and the growing bud and cytokinesis contractile ring in yeast (47). Furthermore, several proteins have been
identified that bind calmodulin in the absence of Ca2+,
such as neuromodulin (48) and the unconventional myosins (reviewed in
Ref. 12). The notion that calmodulin-carrying myosins may play a role
in the determination of the subcellular localization of calmodulin has
been suggested (22, 46, 49). Porter and collaborators (22) even
suggested that calmodulin could translocate from myosin to other
proteins as part of a mechanism of enzyme regulation.
In this paper, we present biochemical evidence that BM-V, an
unconventional myosin which harbors at least 8 calmodulins on its neck
domains, binds to CaMKII and activates its kinase activity in the
presence of Ca2+. Using a combination of
immunoprecipitation and Western blot overlay techniques, we showed
binding (i) between the native proteins for both phosphorylated and
non-phosphorylated species; (ii) between denatured-renatured (Western
blotted) polypeptides, corresponding to the heavy chain of BM-V and the
The question is whether an interaction between CaMKII and BM-V is
indeed plausible based on what is known about the subcellular locations
and functions of these two proteins. In a recent report, BM-V was shown
to be associated with synaptic vesicles via the synaptobrevin-synaptophysin complex (7). These authors suggested that
BM-V may be involved in the recruitment of synaptic vesicles to the
pre-synaptic membrane. We also have detected BM-V in synaptic vesicle
preparations,3 although we
demonstrated here its coimmunoprecipitation with syntaxin, an integral
membrane protein of the synaptic plasma membrane, from detergent
extracts of synaptosomes. Integral membrane proteins of synaptic
vesicles and the synaptic plasma membrane, together with associated and
linking proteins, form multimeric complexes during different stages of
the lifecycle of synaptic vesicles (reviewed in Refs. 34 and 38), which
resist detergent extractions (50). Specifically, syntaxin and SNAP-25
tightly bind synaptobrevin as a priming event for vesicle fusion to the plasma membrane (51), although this complex seems to require the
dissociation of synaptophysin (52). In accordance, we did not detect
synaptophysin in the immunoprecipitate with BM-V and syntaxin (Fig. 2).
Thus, the present data on BM-V associations are not necessarily in
conflict. Clearly, further studies on the subfractionation of
synaptosomes will have to be done to establish the precise
interactions. In any case, the data do support the basic claim that
BM-V is associated with components of the multimeric complexes involved
in the synaptic vesicle exocytotic cycle.
Bi and collaborators (53) have provided evidence for essential roles of
both motor proteins, kinesin and myosin, in the recruitment of vesicles
to the release sites of Ca2+-regulated exocytosis in living
cells. Without identifying the myosin, these studies indicated that a
myosin-mediated step affected Ca2+-regulated exocytosis at
a point upstream from the final fusion event, but downstream from the
kinesin transport event. Importantly, the inhibition of CaMKII had
similar effects at the same stage of exocytosis as did the inhibition
of myosin. A crucial function of synaptic vesicle-bound CaMKII at this
stage of exocytosis is the phosphorylation of synapsin I, which tethers
synaptic vesicles to the actin cytoskeleton in its unphosphorylated
form and releases this interaction when phosphorylated (37).
There is much evidence to suggest that BM-V has a role in vesicle
transport (reviewed in Refs. 10 and 11), including small brain-derived
vesicles containing the synaptic vesicle marker protein, SV2 (54). The
IQ motifs in the neck domain of BM-V have high homology to the
calmodulin-binding sequence of neuromodulin (GAP-43), which has been
suggested to have a role in reversibly sequestering calmodulin at
specific locations near calmodulin-requiring enzymes in the cell (48).
Similarly, calmodulin binds to BM-V at low Ca2+
concentrations and is at least partially released at micromolar Ca2+ (15, 16).
A plausible hypothesis that unites the data we have presented here with
that of others is that, upon elevation of intracellular free
Ca2+, calmodulin could translocate from BM-V to CaMKII,
which in turn would autophosphorylate and then phosphorylate in its
immediate environment synapsin I and BM-V. In the former case, the
tethering effect of synapsin I on synaptic vesicles to the actin
cytoskeleton would be released. In the latter case, we do not know what
effect, if any, phosphorylation of the BM-V tail domain by CaMKII (17) would have, but we can speculate that new ties to the actin
cytoskeleton via BM-V might occur that may participate in the
mobilization of the synaptic vesicles from the "reserve pool" to
the "releasable pool."
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (5000 Ci/mmol) was
purchased from Amersham Pharmacia Biotech and NEN Life Science
Products. Electrophoresis chemicals, molecular mass standards,
imidazole, ATP (grade II), EDTA, EGTA, phenol, glycose, maltose, IPTG,
dithiothreitol, ampicillin, tetracycline, chloramphenicol, aprotinin,
benzamidine, and lysozyme were purchased from Sigma. Nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were obtained from
Sigma or Promega. Anti-rabbit and anti-mouse IgGs conjugated to
alkaline phosphatase were from Promega. Pefabloc was from Roche
Molecular Biochemicals. DNA purification paper (NA4S) was from
Schleicher & Schuell. Klenow, alkaline phosphatase and T4 DNA ligase
were from Life Technologies, Inc. Restriction enzymes were purchased
from Life Technologies, Inc. and New England Biolabs. Taq
polymerase was from Perkin Elmer. Pansorbin cells were from Calbiochem.
Chromatography media were from Amersham Pharmacia Biotech. Grade I
water, prepared using the Milli-Q or Toraypure systems, was used in all solutions.
-subunit of CaMKII and
anti-synaptophysin (Roche Molecular Biochemicals), and anti-syntaxin
and anti-200-kDa neurofilament protein (Sigma). Polyclonal antibodies
against brain myosin II purified from chick brains as described
previously (23) and against chicken brain myosin-V (tail domain
expressed in bacteria, Ref. 13) were generated in rabbits and purified
by affinity to the respective antigen.
20 °C.
-subunit of CaMKII. Fractions enriched in
CaMKII were pooled, supplemented to 1 mM free
Ca2+, and incubated for 1 h at room temperature with
3-5 ml of calmodulin-Sepharose 4B equilibrated in 50 mM
imidazole-HCl, pH 8.0, containing 1 mM CaCl2
and 1 mM DTT. The resin was loaded into a column and washed with 5 volumes of equilibration buffer alone and 2 volumes of equilibration buffer containing 500 mM NaCl, followed by
elution with 50 mM imidazole-HCl, pH 7.4, containing 1.5 mM EGTA. Fractions of 0.5 ml were collected and assayed for
Ca2+/calmodulin-dependent autophosphorylation
activity, or analyzed by dot blots for the
-subunit of CaMKII.
Fractions enriched in CaMKII were pooled, assayed for total protein and
stored at -20 °C in 30% sucrose.
-32P]ATP to a final concentration of
10 µM and stopped by addition of SDS-PAGE sample buffer
containing 1 mM EDTA and boiling, or 2) in experiments
where the protein content was dilute, the reaction was initiated by
addition of 100 µM free Ca2+ immediately
after the addition of [
-32P]ATP and stopped by
addition of EGTA to 2 mM, followed immediately by
precipitation of total protein by addition of 0.10 volume of sodium
deoxycholate, 0.15% w/v, and of 0.15 volumes of ice-cold 50%
trichloroacetic acid. The precipitate was collected by centrifugation at 12,000 × g for 15 min at 4 °C, dissolved in
SDS-PAGE sample buffer, and neutralized by ammonium vapor.
Incorporation of 32P into polypeptides was visualized by
autoradiography of dried gels after SDS-PAGE and quantified by either
of two methods. (a) Appropriate bands corresponding to the
and
subunits of CaMKII were cut from the gel, solubilized in
0.5 ml of 30 volume H2O2 by heating at 80 °C
for 2 h, and quantified by Cerenkov scintillation counting in 5 ml
of H2O. (b) The dried gels were exposed on a storage
phosphor screen, which was subsequently scanned using the STORM 840 PhosphorImager (Molecular Dynamics). Quantitative analysis on
appropriate bands was performed using the ImageQuant software. In
parallel determinations, both methods gave equivalent results, the
STORM system being much quicker and easier.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Myosins II and V are present in
synaptosomes. The crude "mitochondrial" pellet (P2)
obtained from rat cerebral cortex was applied to a discontinuous
Percoll gradient and the resulting visible bands which form at the
interphases (7.5, 10, and 20%
Percoll) and final pellet (P) were collected.
Equivalent amounts of protein from aliquots of these fractions were
subjected to SDS-PAGE and either stained with Coomassie Blue
(SDS-PAGE) or blotted onto nitrocellulose and probed with
specific antibodies, as indicated (Immunoblot). The
positions of molecular mass standards are indicated to the
left of the stained gel. Appropriate positions from the blot
were cut and probed with the corresponding antibodies (see
"Experimental Procedures"): hcBMII, heavy chain of brain
myosin II; hcBM-V, heavy chain of brain myosin-V;
NF200, neurofilament protein; CKII,
-subunit of CaMKII; Synp, synaptophysin. The
arrows indicate the relative positions of the probed
proteins in the gel (note that the immunoblot is a composite of three
blots from this overlapping region of the gel).
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Fig. 2.
CaMKII and syntaxin coimmunoprecipitate with
BM-V from Triton X-100-solubilized synaptosomes. Synaptosomes were
lysed in 9 volumes of 5 mM HEPES, pH 7.5, 1 mM
EGTA, 2 mM DTT, 5 mM MgCl2, 0.3 mM PMSF, 2 µg/ml aprotinin, and 1 mM
benzamidine, containing 1% Triton X-100 and 5 mM ATP, and
centrifuged at 10,000 × g for 20 min. The supernatant
was incubated with Pansorbin cells laden with either BM-V tail
antibodies (anti-BM-V) or pre-adsorbed anti-serum
(control), as described under "Experimental Procedures."
Western blots were prepared from the first supernatant (s)
and immunoprecipitated fraction (p), and appropriate
positions from the blot were cut and probed with the corresponding
antibodies: hcBM-V, myosin-V heavy chain;
CKII,
-subunit of CaMKII; Synp,
synaptophysin; Synt, syntaxin.
CaMKII (the slower migrating band recognized
by the
CaMKII antibody seen only in lane 3p)
were detected. Controls demonstrated that the precipitation of CaMKII in this assay was dependent on the presence of both BM-V and the BM-V
tail antibodies. Similarly, CaMKII coimmunoprecipitated with the
bacterially expressed tail domain (Fig. 3B). Under
conditions where autophosphorylation of CaMKII was promoted (reaction
4, note the requirement for calmodulin), it appeared that the
phosphorylated
-subunit (based on electrophoretic mobility) was the
principal component that coimmunoprecipitated with the tail domain
(lane 4p).
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Fig. 3.
Purified CaMKII coimmunoprecipitates with
purified BM-V or with bacterially expressed BM-V tail domain.
A, BM-V (4 µg) and/or CaMKII (1 µg) were incubated
during 10 min at 37 °C in 170 µl of 25 mM
imidazole-HCl, pH 7.4, containing 1 mM MgCl2,
100 µM Ca2+ and, where indicated, 10 µM ATP. The reaction mix was then diluted to 1 ml with
the same buffer, containing 1 mM ATP and 600 mM
NaCl, and submitted to immunoprecipitation. Immunoblots were prepared
from the first supernatant and immunoprecipitated fraction
(s and p, see "Experimental Procedures") from
reactions containing BM-V alone (1 and 5), BM-V + CaMKII (2 and 6), BM-V + CaMKII + ATP
(3), or CaMKII alone (4). Pansorbin cells laden
with BM-V tail antibodies were used in experiments 1-4, whereas in
experiments 5 and 6 anti-serum preadsorbed with excess BM-V was used as
the control. The appropriate regions of the immunoblots were probed
with tail antibodies (hcBM-V) and a monoclonal antibody
against the -subunit of CaMKII (
CKII), respectively.
Note that the autophosphorylation of the
-subunit of CaMKII results
in multiple phosphorylated species which migrate slower in SDS-PAGE
(lane 3) (26). B, basically the same
experiment as in A except that BM-V was replaced by 50 µl
of bacterial extract of E. coli expressing the BM-V whole
tail domain (clone 8c pET, see "Experimental Procedures").
Immunoblots were prepared from the first supernatant (s) and
immunoprecipitated fraction (p), as above, from reactions
containing tail alone (1 and 6), tail + CaMKII
(2 and 7), tail + CaMKII + ATP (3),
tail + CaMKII + 1.5 µM CaM + ATP (4), and
CaMKII alone (5). Pansorbin cells laden with BM-V tail
antibodies were used in experiments 1-5, whereas in experiments 6 and
7 anti-serum preadsorbed with excess BM-V was used as the control. The
appropriate regions of the immunoblots were probed with tail antibodies
(Tail) and a monoclonal antibody against the
-subunit of
CaMKII (
CKII), respectively.
-32P]ATP. The reaction was then
diluted to 3 ml containing 1 mM non-radioactive ATP and
immunoprecipitation performed as described above. Autoradiography of
the immunoprecipitated proteins separated on a SDS-PAGE gel (Fig.
4) demonstrated that (a) the
- and
-subunits of CaMKII were autophosphorylated and BM-V
phosphorylated, and (b) the phosphorylated species were
coimmunoprecipitated by tail antibodies. The relative intensity of the
bands was not affected by 4-fold dilution of substrate and enzyme in
the kinase reaction, suggesting that the phosphorylated species were
effectively bound during the reaction and fully immunoprecipitated
under these dilute conditions.
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Fig. 4.
Phosphorylated BM-V and
autophosphorylated - and
-subunits of CaMKII coimmunoprecipitate.
Mixtures of 1 µg of BM-V and 1 µg of CaMKII in 345 µl of 25 mM imidazole-HCl, pH 7.4, containing 1 mM
MgCl2 were incubated for 10 min at room temperature and
incubated as such (1) or diluted to 1380 µl
(2), followed by 1 min at 37 °C with 10 µM
[
-32P]ATP (1.5 µCi) and 100 µM
Ca2+. The reactions were then diluted to 3 ml with reaction
buffer containing 1 mM cold ATP and incubated at room
temperature for 50 min. Immunoprecipitation with tail antibody was
performed as described in Fig. 3 in the presence of 600 mM
NaCl, and the first supernatant (s) and immunoprecipitated
pellet (p) were analyzed by autoradiography after SDS-PAGE.
The positions of the heavy chain of BM-V (hcBM-V) and the
and
subunits of CaMKII (CKII) are indicated.
and
Subunits of CaMKII--
To
further characterize the binding between BM-V and CaMKII, we used the
"overlay" technique where Western blots of protein samples were
incubated with protein ligand, followed by detection of binding by
ligand-specific antibodies. When a Western blot of purified BM-V or of
the MBP-tail fusion protein expressed in bacteria was incubated with
purified CaMKII, its binding to the BM-V heavy chain and to the tail
domain was detected by the CaMKII monoclonal antibody (Fig.
5, lanes 2 and
5). Reciprocally, when a blot of CaMKII was incubated with
purified BM-V, both
and
subunits of CaMKII were detected by
BM-V tail antibodies (Fig. 5, lane 8). These data
strongly suggest that there is direct binding between these two
proteins.
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Fig. 5.
The reciprocal binding of BM-V and CaMKII is
shown by an "overlay" assay. Western blots of BM-V, MBP-Tail
purified on an amylose column and CaMKII (CKII) were
incubated either with CaMKII (lanes 2 and 5), with BM-V
(lane 8) or with blocking solution only
(lanes 1, 3, 4,
6, 7, and 9) as described under
"Experimental Procedures." Lane 10 is a
Coomassie Blue-stained gel of the purified CaMKII used in these
studies. The blots were probed with a monoclonal antibody against the
-subunit of CaMKII (lanes 1, 2,
4, 5, and 9) or with BM-V tail
antibodies (lanes 3, 6, 7,
and 8). The positions of BM-V heavy chain
(hcBM-V), undegraded fusion protein (MBP-Tail)
and
and
subunits of CaMKII (CKII) are indicated by
arrowheads to the right of the figure.
CaMKII, followed by enzymatic detection (Fig.
6C). The result demonstrated that autophosphorylated CaMKII
bound much more strongly than non-phosphorylated CaMKII.
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Fig. 6.
Determination of the apparent dissociation
constant for CaMKII binding to BM-V. A, pieces
containing equal amounts of the BM-V whole tail domain, expressed in
bacteria without fusion with MBP, were cut from Western blots and
incubated with increasing concentrations of
32P-autophosphorylated CaMKII, as indicated, in 380 µl of
reaction mix containing 25 mM imidazole, pH 7.0, 2 mM EGTA, 12.5 µg/ml calmodulin, 4 mM
MgCl2, and 1 mM ATP. Blots were washed and
air-dried, exposed on a storage phosphor screen, and imaged using the
STORM 840 PhosphorImager. B, the data were quantitated using
the ImageQuant software by defining a rectangle around the appropriate
band and a second rectangle of equal area immediately above the band to
subtract as nonspecific binding. The apparent dissociation constant was
determined from a double-reciprocal plot (inset), since the
amount of CaMKII bound was less than 1% of the total CaMKII at each
concentration. Four equivalent experiments gave Kd
values that ranged from 1.1 to 7.7 nM. C, direct
comparison of the binding of non-phosphorylated versus
phosphorylated CaMKII to the blotted tail is demonstrated in an
equivalent experiment done by overlaying with CaMKII, followed by
probing the blots with anti- CaMKII and enzymatic detection as in
Fig. 5.
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Fig. 7.
Mapping of the CaMKII binding site(s) on BM-V
heavy chain by the overlay assay. In A, recombinant
proteins corresponding to defined segments of BM-V were expressed in
bacteria, as described under "Experimental Procedures." Samples
were analyzed by Coomassie Blue staining of SDS-PAGE gels, and Western
blots of these bacterial extracts were incubated with
autophosphorylated [32P]CaMKII. CaMKII-binding regions
were revealed by autoradiography (overlay). Lane
1, non-induced bacterial extract; lane
2, bacterial extract expressing MBP alone; lane
3, bacterial extract expressing GST alone. The other lanes
contain the expressed recombinant proteins, as follows: lane
4, head domain, aa 5-752, in fusion with MBP;
lane 5, proximal tail region, aa 911-1122, in
fusion with MBP; lane 6, medial tail region, aa
1117-1435, in fusion with GST; lane 7, globular
tail region, aa 1440-1830, in fusion with GST; lane
8, complete tail domain, aa 899-1830, in the pET5a vector.
The positions of the recombinant proteins in the bacterial extracts
after induction by IPTG are indicated by arrowheads. In
B a linear model of the BM-V molecule and recombinant
proteins is presented with relative scoring of CaMKII binding from the
overlay data, as illustrated in A. The heavy chain of BM-V
(hcBM-V) is divided into head (aa 1-765), neck (aa
766-912), and tail (aa 913-1830), which is further divided into
proximal tail (aa 913-1106), medial tail (aa 1107-1420), which
includes the PEST sequence, and globular tail (aa 1421-1830). The six
IQ motifs are indicated by stripes, and the
cross-hatching in the proximal and medial tail segments
indicate coiled-coil structures. The recombinant proteins, represented
by bars with N- and C-terminal amino acids indicated, are
aligned below their positions on the linear structure. The MBP and GST
fusion counterparts are represented at the N termini by
unconnected and fused rectangles,
respectively.
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Fig. 8.
Protein kinase activity of CaMKII is
activated by BM-V and Ca2+ without requirement for
additional calmodulin. Reaction mixtures (55 µl) contained 25 mM imidazole-HCl, pH 7.4, 4 mM
MgCl2, 1 mM DTT, 1 mM EGTA, 2.5 µg of BM-V, 0.25 µg of CaMKII, and, where indicated, 100 µM free Ca2+ and 50 µM TFP. The
reactions were initiated by the addition of 2.4 µCi of
[ -32P]ATP and stopped after 2 min at 35 °C with
SDS-PAGE sample buffer. The experiments were: 1, BM-V alone;
2, CaMKII alone; 3, BM-V + CaMKII, no
Ca2+; 4, BM-V + CaMKII + Ca2+;
5, CaMKII alone + Ca2+; 6, BM-V + CaMKII + Ca2+ + TFP. Samples of the reactions were analyzed
by SDS-PAGE and autoradiography of the dried gel (Autorad.).
A Western blot of an equivalent gel was probed with BM-V tail
antibodies and with monoclonal antibodies against the
subunit of
CaMKII (Blot) to illustrate the protein content of the
reactions. The heavy chain of BM-V (hcBM-V) and CaMKII
(CKII,
- and
-subunits) are indicated to the
right of the figure. Ca2+-activated protein
kinase activity was not detected in the BM-V fraction (data not
shown).
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Fig. 9.
The time course of autophosphorylation of
CaMKII activated by BM-V suggests an initial burst phase. Reaction
mixtures containing 15 nM BM-V + 2 nM CaMKII + Ca2+, as described in Fig. 8, were initiated by the
addition of 2.4 µCi of [ -32P]ATP, allowed to proceed
for the various times indicated on the abscissa, and stopped
by trichloroacetic acid/deoxycholate precipitation as described under
"Experimental Procedures." Samples were applied to SDS-PAGE gels,
bands corresponding to the
and
subunits of CaMKII were cut out
and dissolved by H2O2, and the incorporation of
32P was determined by Cerenkov counting.
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Fig. 10.
BM-V activates the autophosphorylation of
CaMKII with high affinity. The incorporation of 32P
into CaMKII after 15 s was determined with increasing
concentrations of BM-V or calmodulin purified from bovine brain. The
concentration of CaMKII was fixed at 3.3 nM in
imidazole-HCl, pH 7.4, containing 4 mM MgCl2, 1 mM EGTA, and 1 mM DTT. BM-V or calmodulin at
the indicated concentrations was added to the reaction mix and
incubated for 1 h at room temperature. 2.4 µCi of
[ -32P]ATP followed by 100 µM free
Ca2+ were added to initiate the reaction, which was stopped
after 15 s by trichloroacetic acid precipitation, as described
under "Experimental Procedures." The incorporation of
32P into CaMKII was determined after SDS-PAGE by exposure
of the dried gels to a storage phosphor screen and quantitated by the
STORM system as described in Fig. 6. A, the data are plotted
against the molar concentration of the respective activating proteins.
B, double-reciprocal plots of the same data gave apparent
affinity constants of 10.5 and 26.5 nM for BM-V and
calmodulin, respectively. The range for four independent determinations
was 10-19 nM and 12-52 nM for BM-V and
calmodulin, respectively. The maximum incorporation of 32P
into CaMKII activated by BM-V was about twice that activated by
calmodulin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits of CaMKII, to the appropriate alternate native
protein; and (iii) between autophosphorylated CaMKII and bacterially
expressed proteins corresponding to the whole tail, medial tail, and
proximal tail regions of the BM-V molecule, but not to the head domain
nor to the C-terminal globular tail domain. The binding was resistant
to 600 mM NaCl and 1% Triton X-100, and under the
conditions measured (Western blots of the bacterially expressed tail
domain overlaid with autophosphorylated CaMKII) was of relatively high
affinity. The binding of non-phosphorylated CaMKII to the tail domain
was also detected, although very much weaker and not quantifiable with
our methods. We also demonstrated that BM-V was able to activate the
kinase activities of CaMKII in a
Ca2+-dependent, trifluoperazine-inhibited
manner without the need for additional calmodulin. Comparison between
the apparent activation constants for BM-V versus calmodulin
suggested that each BM-V provided 2 or more calmodulins. Interestingly,
activation of the autophosphorylation of CaMKII by BM-V resulted in a
higher maximum level of incorporation of 32P than that by
purified calmodulin. Our data suggested that this activation included
an initial burst of autophosphorylation, but our experimental protocol
did not allow us to distinguish this burst from the initial
steady-state rate of autophosphorylation. However, this result did
suggest that BM-V not only supplies calmodulin to CaMKII, but that its
binding alters the kinetic properties or autophosphorylation sites of
CaMKII. These biochemical results obtained in vitro are
consistent with the possibility that these two proteins interact in the
intracellular milieu.
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ACKNOWLEDGEMENTS |
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We thank Domingus E. Pitta, Silmara R. Banzi, and Silvia Regina Andrade for expert technical assistance and Benedita O. de Souza for auxiliary help.
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FOOTNOTES |
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* This work was supported in part by Fundação de Amparo à Pesquisa do Estado de São Paulo Grant 93/3552-9, Programa de Apoio ao Desenvolvimento Científico e Tecnológico Grant 62.0099/95.0, Conselho Nacional de Desenvolvimento Científico e Tecnológico Grant 522791/95-6, and the Comissão de Cooperação Internacional of the University of Sao Paulo (all to R. E. L. and E. M. E.).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.
§ Predoctoral fellows supported by Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior.
Recipient of an undergraduate research stipend from Conselho
Nacional de Desenvolvimento Científico e Tecnológico.
** To whom correspondence should be addressed: Dept. of Biochemistry, Faculdade de Medicina de Ribeirão Preto, Av. Bandeirantes, 3900, Ribeirão Preto, Sao Paulo 14049-900 Brazil. Tel.: 55-16-602-3319; Fax: 55-16-633-6840; E-mail: relarson{at}fmrp.usp.br.
2 F. S. Espindola, D. M. Suter, R. E. Cheney, S. M. King, and M. S. Mooseker, manuscript in preparation.
3 F. Mani, E. M. Espreafico, and R. E. Larson, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
BM-V, brain
myosin-V;
aa, amino acid(s);
DTT, dithiothreitol;
TFP, trifluoperazine;
IPTG, isopropyl-1-thio-- D-galactopyranoside;
MBP, maltose-binding protein;
GST, glutathione S-transferase;
CaMKII, calmodulin-dependent protein kinase II;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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