From the Department of Chemistry, Graduate School of
Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan, ¶ Integrated Proteomics System Project, Pioneer Research on
Genome the Frontier, Ministry of Education, Culture, Sports, Science
and Technology, Department of Chemistry, Graduate School of Science,
Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan,
Department of Biology, Faculty of Science, Chiba University,
Chiba 263-8522, Japan, and ** Division of Proteomics
Research (ABJ-Millipore), The Institute of Medical Science, The
University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
Received for publication, November 12, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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V-1 is a 12-kDa protein consisting of three
consecutive ANK repeats, which are believed to serve as the surface for
protein-protein interactions. It is thought to have a role in neural
development for its temporal profile of expression during murine
cerebellar development, but its precise role remains unknown. Here we
applied the proteomic approach to search for protein targets that
interact with V-1. The V-1 cDNA attached with a tandem affinity
purification tag was expressed in the cultured 293T cells, and the
protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the The V-1 protein was originally identified in the murine cerebellum
as one of the proteins expressed significantly at the initial stage of
postnatal development (1), particularly in the regions where synaptic
formation and neuronal migration occur actively during neurogenesis (2,
3). V-1 consists of 117 amino acids containing three contiguous repeats
of the ANK motif, alternatively called the cdc10/SWI6 motif (1, 2),
which is crucial for a large number of protein-protein interactions
(4). Previous studies suggested the potential role of V-1 in the signal
transduction pathways leading to catecholamine synthesis or to cardiac
hypertrophy. For example, Yamakuni et al. (5-7) demonstrate
that the overexpression of V-1 caused a significant increase in the
catecholamine level in PC12 cells, presumably through the
transcriptional activation of the genes for catecholamine synthesis. In
other reports (8-11), V-1 was designated as "myotrophin" and was
shown to participate in the cell signaling pathways for the
NFkB-mediated activation of protein synthesis in the myocytes. Thus,
both of these studies (5, 9) suggested the roles of V-1 in the
biological events taking place in the nucleus. However, no biological
function has been attributed to V-1 in the cytoplasm in which this
molecule predominantly resides within the cells and tissues (5, 9).
In this study, we screened for V-1-binding proteins by a novel
proteomic approach that combined the tandem affinity purification (TAP)1 procedure (12) and
mass spectrometry (MS). Following this strategy, we identified capping
protein (CP) as a V-1-binding protein. We confirmed the existence of
the V-1·CP complex not only in cultured cells transfected with the
TAP-tagged V-1 but also endogenously in cells and rat cerebellar
extracts. Furthermore, we found that V-1 inhibited the CP-regulated
actin polymerization. On the basis of these results, the possible role
of V-1 in neuronal development is discussed.
Materials--
Actin monomer was prepared from the
acetone-dried powder of rabbit skeletal muscle according to the
procedure of Spudich and Watt (13). Pyrene-labeled actin was purchased
from Cytoskeleton Inc. (Denver, CO). The polyclonal antibody against
the V-1 protein was raised in rabbits injected with the recombinant V-1
protein. The antibody was purified by ammonium sulfate fractionation
(0-50% saturation) followed by affinity chromatography on
Sepharose beads coupled with the recombinant V-1 protein. The anti-CP
monoclonal antibody (14) was obtained from the Developmental Studies
Hybridoma Bank, developed under the auspices of the National Institute
of Child Health and Human Development, and maintained by The University of Iowa, Department of Biological Sciences. Oligonucleotides were purchased from Sigma.
Plasmid Constructions--
To construct the cDNA for
TAP-tagged V-1, the TAP cDNA (pBS1479) (12) was first digested with
BamHI and HindIII and was inserted into the
BamHI and EcoRI sites of the mammalian expression vector pcDNA3 (Invitrogen) after blunting the HindIII
and EcoRI sites (termed pcDNA3-TAP). The V-1 cDNA
was then generated by PCR using the oligonucleotides
5'-GCGAAGCTTATGTGCGACAAGGAGTTCAT-3' and
5'-CTGGATCCCTGGAGAAGAGCTTTGATTG-3' and the rat V-1 cDNA (2). The PCR fragment was digested with HindIII and
BamHI and was inserted into the cloning site of
pcDNA3-TAP.
The cDNA for GST-tagged V-1 was constructed by PCR amplification of
the V-1 cDNA using the oligonucleotides
5'-TAGGATCCTGTGCGACAAGGAGTTCATG-3' and
5'-GCGAATTCTCACTGGAGAAGAGCTTTGA-3' and the rat V-1 cDNA. The PCR
fragment was digested with BamHI and EcoRI and
was inserted into the cloning sites of the bacterial expression vector
pGEX-3X (Amersham Biosciences). The V-1 cDNA was also amplified by
PCR using the oligonucleotides
5'-TACCATGGGATGTGATAAAGAGTTCATG-3' and
5'-GTTCATGGATCCATCACTGGAGAAGAGC-3' and the rat V-1 cDNA. The resulting fragment was digested with NdeI and
BamHI and was inserted into the cloning sites of the
bacterial expression vector, pET 15b (Novagen, Madison, WI).
Cell Culture and Immunoprecipitation--
Human embryonic kidney
293 T cells (293T) were maintained in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum (Invitrogen) as described
previously (15). The cells (2 × 106 cells) were
cultured in a 100-mm dish and were transfected the following day with 8 µg of cDNA using the Polyfect transfection reagent (Qiagen,
Hilden, Germany) according to the manufacturer's instructions. After
48 h, the cells were washed twice with phosphate-buffered saline
and were immediately scraped into 500 µl of lysis buffer containing
50 mM Tris-HCl (pH 8.0), 10% glycerol, 1% Triton X-100, 150 mM NaCl, 100 mM NaF, 5 µM
ZnCl2, 1 mM Na3VO4, 10 mM EGTA, 2 µg/ml leupeptin, and 4 mM
phenylmethylsulfonyl fluoride. The cell lysate was centrifuged
at 100,000 × g for 20 min at 4 °C, and the
supernatant (~5-mg protein) was incubated with 20 µl of IgG-Sepharose beads (Amersham Biosciences) for 2 h at 4 °C. The IgG beads were washed five times with 500 µl of a buffer containing 20 mM Tris-HCl (pH 7.5), 5% glycerol, 0.1% Triton X-100,
and 150 mM NaCl and twice with 50 mM Tris-HCl
(pH 8.0). The beads were then mixed with tobacco etch virus
protease (Invitrogen) at room temperature for 1 h in 50 mM Tris-HCl (pH8.0) to release the protein complex into
solution. The complex was subsequently incubated with 20 µl of
calmodulin-agarose beads (Stratagene, La Jolla, CA) in a buffer
containing 50 mM Tris-HCl (pH 8.0), 150 mM
NaCl, and 1 mM CaCl2. After washing the beads
twice with the same buffer, the bound proteins were eluted from the
beads with this buffer containing 4 mM EDTA instead of
1 mM CaCl2.
In-gel Digestion and Tandem Mass Spectrometry--
Protein bands
(0.2 × 0.8 cm) were excised from the SDS gel, dehydrated with
acetonitrile, and vacuum-dried. The gel pieces were swollen in 10 µl
of 100 mM Tris-HCl (pH 8.8), and the proteins were in-gel
digested overnight with 250 ng of trypsin (Promega, Madison, WI) at
37 °C. The peptide fragments were extracted from the gel with 100 µl of 50% acetonitrile in 5% formic acid and were concentrated by
adsorption onto 0.1 µl of reversed-phase beads (POROS R2, Applied
Biosystems, Foster, CA). After the beads were washed twice with 400 µl of 0.1% trifluoroacetic acid, the bound peptides were recovered
with 2 µl of 50% methanol in 5% acetic acid. The peptide mixture
was then analyzed by tandem mass spectrometry on a electrospray
ionization quadrupole time-of-flight mass spectrometer (Q-TOF2,
Micromass, Manchester, United Kingdom) equipped with a nanospray tip.
All of the MS/MS spectra were searched against the non-redundant
protein sequence data base maintained at the National Center for
Biotechnology Information to identify proteins by the Mascot program
(Matrixscience, London, United Kingdom). The MS/MS signal assignments
were confirmed manually.
Expression and Purification of Recombinant
Proteins--
Recombinant V-1 protein was expressed in
Escherichia coli (BL21) and was purified by ammonium sulfate
fractionation (50-85% saturation) followed by anion exchange
chromatography on a DEAE-5PW column (0.75 × 15 cm, TOSOH, Tokyo,
Japan). The GST-tagged V-1 protein (GST-V-1) was purified with
glutathione-Sepharose beads (Amersham Biosciences) as described
previously (16). The CP heterodimer consisting of Surface Plasmon Resonance Analysis--
All of the studies were
performed on a Biacore 2000 instrument (Biacore, Uppsala, Sweden). The
GST or GST-fused V-1 protein was immobilized on the CM5 sensor chip via
an anti-GST antibody (Biacore). The concentration of the immobilized
protein was adjusted to yield ~180 resonance units. The V-1/CP
interaction analysis was carried out at 25 °C by introducing CP Assay of CP-mediated Actin Nucleation and
Depolymerization--
The CP-mediated actin nucleation and
depolymerization were analyzed essentially according to the procedures
described by Higashi and Oosawa (19) and Schafer et al.
(14), respectively. For the actin nucleation assay, 4.8 µM actin monomer and 0.48 µM CP were
incubated in a G-actin buffer (0.1 mM CaCl2,
0.2 mM ATP, 2 mM HEPES-KOH (pH7.0)) in the
presence or absence of 2.4 µM V-1 for 10 min at room
temperature, and the polymerization was initiated in 50 mM
KCl, 1 mM MgCl2, and 10 mM
HEPES-KOH (pH 7.0). The actin nucleation was assayed as the initial
rate of increase in the absorbance at 237 nm. For the actin
depolymerization assay, the F-actin was prepared by polymerizing
4.8 µM actin (50% Pyrene-labeled) for 90 min at 25 °C
in F-actin buffer (2 mM MgCl2, 100 mM KCl, 0.1 mM CaCl2, 0.2 mM ATP, 10 mM HEPES-KOH (pH7.0)). The
depolymerization was initiated by the dilution of 25 µl of F-actin
solution with 1.2 ml of G-actin buffer containing 10 µg/ml human
serum albumin, 10 nM CP, and various amounts of recombinant
V-1. The depolymerization of actin filaments was assayed by monitoring
the changes in fluorescence at 407 nm of the Pyrene-labeled actin with
an excitation wavelength at 365 nm.
F-actin Co-sedimentation Assay and Other Analytical
Methods--
Actin monomer (4.8 µM) was polymerized for
90 min at 25 °C in the F-actin buffer in the presence of CP (3 µM) with or without 15 µM V-1 protein. The
polymerized mixture was centrifuged for 30 min at 100,000 × g, and the precipitates were analyzed by SDS-PAGE (CBB
staining). SDS-PAGE and Western blotting were carried out as described
previously (20). Silver staining was performed without glutaraldehyde
(21).
Identification of CP as a V-1-binding Protein--
To search for
the V-1-binding protein, we carried out an affinity purification
experiment using the TAP method (12). The TAP method utilizes two
affinity modules (a calmodulin-binding peptide and a protein A epitope)
separated by a cleavage site for the tobacco etch virus protease.
Therefore, the purification produces much less background than the
conventional single epitope tags such as Myc or hemagglutinin.
To adapt this method for our purpose, we constructed a mammalian
expression vector encoding V-1 with a carboxyl-terminal TAP tag,
transiently transformed 293T cells with the vector, and purified the
tagged V-1 with its binding proteins from the cell lysate by the TAP
method. Fig. 1A, lane
2, shows the SDS-PAGE photographs of the purified protein complex.
In addition to the tagged V-1 used as bait, the 36- and 33-kDa
polypeptide bands were reproducibly detected in the cells transfected
with a TAP-tagged V-1 but not in the control cells (Fig. 1A,
lane 1), suggesting that these polypeptides were associated with the expressed V-1 protein. These bands (assigned as bands 1 and 2 in Fig. 1A) were excised from the
gel and subjected to in-gel tryptic digestion, and the peptide
fragments thus generated were analyzed by nanoelectrospray tandem
mass spectrometry, respectively.
Two doubly charged peptide ions with m/z 599.40 and 786.00, were observed from band 1. The data base
analysis of the MS/MS spectrum of the peptide ion with
m/z 599.40 showed that it corresponded to the
sequence LLLNNDNLLR of CP Detection of the Endogenous V-1·CP Complex--
The
overexpression of a particular protein sometimes induces artificial
interactions among proteins, which do not occur under physiological
conditions. To exclude the possibility that the observed interaction
between V-1 and CP might be artificial, we sought to detect the
endogenous V-1·CP complex in cultured 293T cells as well as in rat
cerebella. Soluble extracts were prepared from 293T cells or from rat
cerebella at a postnatal day 12, and the V-1 protein was
immunoprecipitated from the extracts with the anti-V-1 IgG immobilized
on Sepharose beads. The precipitate was then analyzed by SDS-PAGE
followed by immunoblotting with the antibody against the CP Biochemical Properties of the Interaction between V-1 and
CP--
To study the biochemical characteristics of the V-1·CP
complex in further detail, we prepared a number of materials including V-1, GST-tagged V-1, and the CP
To clarify the formation of the V-1·CP complex and to determine the
molecular stoichiometry, the "native-PAGE assay" (24) was performed
(Fig. 3D). When the recombinant V-1 was mixed with CP under
physiological conditions and was analyzed by gel electrophoresis without chaotropic reagents, a new protein band appeared that migrated
between the V-1 and CP heterodimer (Fig. 3D, left
panel). The subsequent analysis of this protein band by SDS-PAGE
revealed that it was a V-1·CP complex, because it contained V-1 and
the CP Suppression of CP Activity by V-1--
CP nucleates actin
polymerization and caps the barbed ends of actin filaments (14, 23, 25,
26). To examine the effects of V-1 protein on the activities of CP, we
assayed the initial rates of polymerization with the CP-nucleated actin
and depolymerization of the CP-capped F-actin in the presence and
absence of the V-1 protein. For the CP-nucleated actin polymerization,
we monitored the changes in optical absorption to measure the initial
rate of actin polymerization. As shown in Fig.
4Aa, CP markedly increased the
initial rate of actin polymerization and the V-1 protein canceled this
CP function. The difference in the initial rate of polymerization between CP-nucleated actin in the presence and absence of the V-1
protein was statistically significant (Fig. 4Ab). The V-1 protein reduced the nucleation activity of CP in a
dose-dependent manner, whereas V-1 alone had no effect on
the actin polymerization (data not shown). Besides this activity, we
also measured the effects of V-1 on the dilution-induced
depolymerization of the CP-capped actin filaments by monitoring the
changes in fluorescence (Fig. 4B). After the preincubation
with V-1, CP reduced its capping activity to depolymerize the actin
filaments. Thus, the capping activity of CP was dependent on the
concentration of V-1, whereas V-1 alone had no effect on the actin
depolymerization. These results demonstrate that the V-1 protein
suppressed the CP activities of both actin nucleation and F-actin
capping.
Interaction between the V-1·CP Complex and F-actin--
We
studied whether the V-1·CP complex bound F-actin by using a
co-sedimentation assay. The actin monomer was polymerized and co-sedimented with CP in the presence or absence of the V-1 protein, and aliquots of the precipitates were analyzed by SDS gel
electrophoresis. As shown in Fig. 5, CP
co-sedimented with F-actin only in the absence of the V-1 protein.
Thus, V-1 appeared to form a stable complex with CP and to prevent CP
binding to F-actin.
CP is one of the F-actin-binding proteins that caps the barbed end
of actin filaments and nucleates the actin polymerization in a
Ca2+-independent (22, 23, 27, 28) and a
phosphatidylinositol 4,5-bisphosphate-dependent manner
(29-32). This activity is thought to be functionally significant,
because the actin-based movement of Dictyostelium is
proportional to the expression level of CP (33) and because CP is
essential for the in vitro reconstitution of the cell
movement (34, 35). In this study, we have shown that the V-1 protein
forms a stable stoichiometric complex with CP in vitro as
well as in vivo (Figs. 2 and 3) and inhibits the CP-mediated
nucleation of actin polymerization. Therefore, we assume that V-1
participates in the regulation of actin dynamics in the cells via the
interaction with CP.
Our strategy to identify the V-1-interacting molecules was based on the
tandem affinity purification of the V-1 complex with a TAP tag followed
by protein identification by mass spectrometry. The TAP method was
originally developed to analyze interactions among yeast proteins (12).
We constructed a mammalian expression vector for the TAP method and
applied it to the mammalian 293T cell line. Even though the V-1 protein
is a rather minor cellular component and a transient expression system
was used for the assay, the method enabled us to isolate a sufficient
amount of the V-1·CP complex for characterization by nanospray tandem
mass spectrometry (Fig. 1). This affinity-tag technique coupled with
mass spectrometry is useful to detect novel protein interactions not
only in yeast but also in mammalian cells.
The V-1 protein consists of three consecutive ANK repeats with an
additional short stretch of sequence (1, 2, 37). The ANK repeat is a
structural motif found in many proteins (36) and mediates specific
interactions with a diverse array of protein targets (4). In the
tertiary structure of V-1 determined by NMR spectroscopy (37), the ANK
repeats comprise the hairpin-helix-loop-helix modules where the Two distinct proteins, carmil (40) and twinfilin (24), are known to
bind CP. Carmil is a scaffold protein containing the CP-binding site,
the myosin I-binding site, the short sequence commonly found in several
actin monomer-binding proteins, and the acidic stretch that can
activate Arp2/3-dependent actin nucleation (40). The
NH2-terminal region of carmil binds CP, but its activity with CP is unknown. Twinfilin is a ubiquitous actin monomer-binding protein composed of two ADF/cofilin-like domains connected by a
short linker region and has a role in the regulation of actin turnover
(41). Twinfilin also forms a stable complex with CP but does not affect
its activity. V-1 is neither a scaffold protein nor an actin-binding
protein, and it lacks structural homology to either carmil or
twinfilin. Thus, V-1 belongs to a novel CP-binding protein category.
Recently, Ena/VASP was reported as an anti-capping molecule, which
promotes actin filament elongation by associating with the barbed ends
of actin and shielding them from CP (42). The actin cytoskeleton in the
Ena/VASP-deficient cell contained shorter, more highly branched
filaments than those in the control cells. V-1 resembles Ena/VASP in
the activity of CP-regulated actin polymerization, but whether a
similar phenotype can be attributed to the V-1-deficient cell is
currently unknown.
Previous studies suggested the potential roles of V-1 in nuclear events
such as the transcriptional activation of a set of enzymes involved in
catecholamine synthesis (5-7) or the regulation of de novo
protein synthesis (8-10, 43). This study suggests for the first time
that V-1 functions in the molecular events taking place in the
cytoplasm in which the major portion of V-1 resides within the cells as
revealed by previous immunohistochemical studies (5, 9). V-1 also
appeared to be a typical cytoplasmic protein in terms of amino acid
sequence with no apparent nuclear localization signal. Our previous
studies revealed the characteristic temporal profile of V-1 expression
in the developing murine cerebellum at postnatal days 7-12 (2).
Namely, V-1 expression is particularly significant during the migration
of progenitor granule cells from the external to internal granular
layer to make synaptic contacts with the target Purkinje cells.
Likewise, the dynamics of actin polymerization play a pivotal role in
the maturation of granule cells, because the modulation of the barbed
ends of actin filaments with cytochalasins changed the behavior of the
growth cone (44) and the migration of granule cells (45). These
observations coupled with the results reported here suggest that V-1
may have a role in the CP-mediated actin-driven cell movements and
motility such as granular cell migration and synapse formation.
and
subunits of the capping protein (CP, alternatively called CapZ or
-actinin). CP regulates actin polymerization by capping the
barbed end of the actin filament. The V-1·CP complex was
detected not only in cultured cells transfected with the V-1 cDNA
but also endogenously in cells as well as in murine cerebellar
extracts. An analysis of the V-1/CP interaction by surface
plasmon resonance spectroscopy showed that V-1 formed a stable complex
with the CP heterodimer with a dissociation constant of 1.2 × 10
7 M and a molecular stoichiometry of
~1:1. In addition, V-1 inhibited the CP-regulated actin
polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that
regulates the dynamics of actin polymerization by interacting with CP
and thereby participates in a variety of cellular processes such as
actin-driven cell movements and motility during neuronal development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits
was produced in E. coli (BL21) using the simultaneous
expression system as described by Soeno et al. (17). For the
experiment shown in Fig. 3, A and B, the
and
subunits were each expressed separately in E. coli
(BL21). Because the expressed
and
subunits were each recovered
in the insoluble fraction, the precipitates were collected and
dissolved in buffer A (2 mM dithiothreitol, 50 mM Tris-HCl (pH 7.5)) containing 8 M urea. The
concentration of urea was then decreased to 0.06 M by a
two-step dilution. First, the solution was diluted with buffer A
containing 6 M urea to a protein concentration of 200 nmol
protein/ml, and the insoluble material was removed by centrifugation.
The soluble fraction then was diluted 100-fold with the surface plasmon
resonance (SPR) buffer (150 mM NaCl, 3 mM EDTA,
0.005% Polysorbate 20, 10 mM HEPES (pH 7.4)). For
the experiment shown in Fig. 3B, the
and
subunits
were mixed and reconstituted to the heterodimer at 4 °C for 10 min.
,
CP
, or the reconstituted CP heterodimer (1 µM) at a
flow rate of 20 µl/min into the Biacore instrument, and the changes
in the SPR signals were recorded. For the kinetic analysis, various
concentrations of the CP heterodimer were loaded on the sensor surface
to equilibrate the V-1/CP interaction, and the concentration of
the complex (Req) was measured as the response units. The correlation
among Req, the concentration of ligand (C) passed over the sensor
surface, and the total binding capacity (Rmax)
of the immobilized protein was: Req/C = KaRmax
KaReq (18). Thus, the association constant
(Ka) was determined from the plot of Req/C
versus Req estimated at different CP concentrations by a
Scatchard plot analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of CP as a V-1-binding
protein. A, the 293T cells were transfected with the
TAP-tagged V-1 construct (lane 2) or the vector alone
(lane 1), and the expressed protein was recovered with the
associating proteins as described under "Experimental Procedures."
The proteins were analyzed by SDS-PAGE (10% polyacrylamide gel, silver
staining). The molecular mass markers are shown on the left,
and the positions of CP (36 kDa) and
(33 kDa) are indicated by
arrows. The asterisk indicates the TAP-tagged V-1
protein. B and C, the doubly charged ions of the
tryptic peptide (m/z = 599.40) from the
36-kDa band (B) and of the tryptic peptide
(m/z = 586.30) from the 33-kDa band
(C) were analyzed by nanoelectrospray ionization-MS/MS. The
amino acid sequences were verified by manual interpretation of the
b-type (italic text) and y-type (normal text)
product ion series as indicated in the figure.
(GenBankTM accession number
P52907) at residues 38-47. The manual assignment of the
fragment ions also yielded the same sequence (Fig. 1B). Likewise, the MS/MS spectrum of the other peptide ion with
m/z 786.00 was assigned to the sequence
FTITPPTAQVVGVLK of CP
at residues 179-193 (data not shown),
confirming that band 1 contained CP
. From band
2, two doubly charged peptide ions with m/z
586.20 and 677.30 were observed. The analysis of their
collision-induced dissociation fragments indicated that one
(m/z 586.20) corresponded to the sequence
STLNEIYFGK of CP
(GenBankTM accession number P47756) at
residues 226-235 (Fig. 1C) and the other
(m/z 677.30) corresponded to the sequence
SGSGTMNLGGSLTR of the same polypeptide at residues 182-195 (data not
shown). The molecular weights of bands 1 and
2 estimated by SDS-PAGE (Fig. 1A) were also
agreed with those of the respective CP subunits (32,902 and 31,331).
Because CP is a stable heterodimer of
and
subunits (22, 23) and
because these polypeptides appear in almost equimolar amounts in the
captured V-1 complex (Fig. 1A), we anticipated that V-1
bound the CP heterodimer itself.
subunit.
As shown in Fig. 2, the CP
was clearly
detected in the precipitates derived both from the 293T cells and the
rat cerebella, suggesting that the V-1·CP complex was present under
physiological conditions in vivo.
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Fig. 2.
Detection of the endogenous V-1·CP
complex. The lysate of 293T cells or the extract of rat cerebella
on postnatal day 14 (~5 mg of protein) was incubated with the
anti-V-1 (V-1) or preimmune (ctrl) antibody. The
resulting immunoprecipitates (IP) were collected on protein
A-Sepharose beads, subjected to SDS-PAGE, and analyzed by
immunoblotting (IB) with the anti-CP antibody
(upper panel) or with the anti-V-1 antibody (lower
panel).
and
subunits. These proteins were expressed in the E. coli cells and were purified to
near homogeneity as observed by SDS-PAGE (Fig.
3A). First, we studied the
complex formation of V-1 and CP by SPR. For the direct binding assay on
the SPR biosensor, the GST-V-1 protein was attached to the sensor
surface via the GST antibody, and the recombinant CP was passed over
the sensor chip. As shown in Fig. 3B, positive binding
signals were detected when the CP heterodimer, reconstituted from the
recombinant CP
and
subunits, was introduced to the sensor chip.
Interestingly, however, the purified CP
or CP
alone gave no SPR
signals (Fig. 3B) even with repeated binding experiments using different subunit preparations, suggesting that V-1 bound specifically to the heterodimeric CP molecule. To obtain kinetic data
of the complex formation, the steady-state resonance was measured using
various concentrations of CP (Fig. 3C). The results showed
that the immobilized V-1 was saturable with respect to the CP binding
and the association constant calculated from the slope of the straight
line of the corresponding Scatchard plot (inset) was
8.4 × 106 M. The Ka
value was not influenced by the amount of immobilized V-1 protein (data
not shown). The equilibrium dissociation constant
(Kd) calculated from 1/Ka was
1.2 × 10
7 M, indicating that the
V-1·CP complex is relatively stable.
View larger version (26K):
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Fig. 3.
Biochemical characterization of the V-1·CP
complex. A, SDS-PAGE profiles of the CP
(lane 1), CP
(lane 2), GST-V-1 (lane
3), and V-1 (lane 4) protein used for the
binding experiments (CBB staining). The molecular mass markers are
indicated on the left. B, analysis of the
interaction of CP and V-1 on the SPR Biosensor (Biacore 2000). The
running buffer was 10 mM HEPES (pH7.4) containing 150 mM NaCl, 3 mM EDTA, and 0.005% Polysorbate 20. The GST-fused V-1 protein was immobilized on a sensor chip, and the SPR
signals were measured to monitor the binding to CP
(
), CP
(
), and the CP heterodimer (
). The resonance unit
(RU), a relative indicator of the protein-protein
interaction, is plotted as a function of time in seconds. C,
steady-state resonance (Req) is plotted at each CP
concentration. Inset, the Scatchard plot derived from the
data in C for CP. The straight line was drawn by
the method of least squares. D, native-PAGE assay.
Left panel, V-1 or CP alone (lanes 1 and
2) or the mixture of V-1 and CP (lane 3) was
analyzed by PAGE on a 4% gel without SDS in 25 mM Tris and
194 mM glycine (pH 9.0). The concentration of V-1 was 2 µM (lane 1) or 20 µM (lane
3), respectively, and the concentration of CP was 2 µM (lanes 2 and 3). Electrophoresis
was performed at 20 mA for 50 min, and the gel was stained with CBB.
Right panel, the stained bands indicated as CP
(lane 2) and Complex (lane 3) were
excised from the gel, destained in 20% MeOH containing 7% AcOH, and
then analyzed by SDS-PAGE (CP, lane 4; complex, lane
5) (CBB staining). The proteins were semi-quantitated by
densitometry performed as described previously (21).
and
subunits (Fig. 3D, right
panel). The semi-quantitative analysis of the CBB-stained bands
indicated that the density of each polypeptide band was 1.0:0.8:1.0 for
CP
,
, and V-1, respectively. This finding suggests that the
binding stoichiometry of the V-1·CP-heterodimer complex is ~1:1,
i.e. one V-1 molecule associates with one CP heterodimer.
V-1 exists in the mouse cerebellum at a concentration of 3.7 × 10
7 M as estimated by quantitative
immunoblotting,2 and this
concentration is almost the same level as that of CP (2
20 × 10
7 M) reported in previous studies (14, 32,
33). Thus, the V-1/CP interaction could occur under
physiological condition in terms of cellular levels of V-1 and CP and
the binding kinetics of these proteins.
View larger version (15K):
[in a new window]
Fig. 4.
Inhibition of CP-mediated actin nucleation
and capping by the V-1 protein. A, effect of V-1 on the
CP-mediated actin nucleation. Actin and the indicated proteins were
incubated for 10 min at room temperature, and the actin polymerization
was initiated by adding the salt solution (50 mM KCl and 1 mM MgCl2) into the mixture. The actin
nucleation was monitored by UV absorption at 237 nm. The time-course
representation (a) and the statistical evaluation
(b) of the V-1 activity on CP-nucleated actin polymerization
are shown. The rate of actin nucleation in b was measured as
the rate of increase in absorbance for the first 100 s. Each value
represents the ratio to the control experiment (b, No
addition) and the mean ± S.E. of three independent
measurements. Asterisks indicate significant difference by
Student's t test (p < 0.005) from CP
(*) and CP+V-1 (**). B, effect of V-1
on the capping activity of CP in the F-actin depolymerization. The
F-actin was prepared as described under "Experimental Procedures,"
and the depolymerization was initiated by dilution with the buffer
containing CP and V-1 at the molar ratios indicated in the figure. The
fluorescence changes in the pyrene-labeled actin versus time
after dilution are shown.
View larger version (32K):
[in a new window]
Fig. 5.
V-1 induced inhibition of the CP/F-actin
interaction. The actin monomer was polymerized in the presence of
CP with (lane 1) or without the V-1 protein (lane
2). After centrifugation, the precipitate (2.5-µg protein) was
analyzed by SDS-PAGE to detect the CP that co-sedimented with the actin
filament (CBB staining).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices lie along one side providing a structural framework and the
hairpins protrude on the other side of the molecule. The hairpins and
the surface of the
helices form a groove-like structure, which is
believed to be responsible for the contact with the target molecule.
This study identified CP as a potential target of V-1. Interestingly,
V-1 bound to the functional CP heterodimer consisting of the
and
subunits but did not bind each of the two subunits. This finding is
comparable with previous observations that each of the CP subunits was
unstable and did not bind actin in vitro and in
vivo (26, 38, 39). Thus, it seems likely that V-1 recognizes the
structural interface of the CP heterodimer by its groove-like ANK
repeats and covers the F-actin binding surface located in the
carboxyl-terminal region of the CP
subunit (26). However, whether
this is the molecular mechanism by which V-1 inhibits the interaction
of CP and F-actin awaits further structural investigation.
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FOOTNOTES |
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* This study was supported in part by grants for the Integrated Proteomics System Project and Pioneer Research on Genome the Frontier from the Ministry of Education, Culture, Sports, Science and Technology of 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 correspondence should be addressed: Dept. of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minami-Osawa 1-1, Hachioji, Tokyo 192-0397, Japan. Fax: 81-426-77-2525; E-mail: mango@comp.metro-u.ac.jp.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M211509200
2 M. Taoka, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathione S-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells.
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