From the Department of Molecular Biology, Graduate
School of Medical Science, Kyushu University, Higashi-ku, Fukuoka
812-8582, § Department of Biology, Graduate School of
Science, Kyushu University, Higashi-ku, Fukuoka 812-8581, ¶ Department of Molecular and Cellular Biology, Laboratory of
Embryonic and Genetic Engineering, Medical Institute of Bioregulation,
Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka
812-8582, and
Division of Protein Metabolism, Institute for
Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka
565-0871, Japan
Received for publication, December 11, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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The vesl-1S/homer-1a gene is
up-regulated during seizure and long term potentiation. Other members
of the Vesl family, Vesl-1L, -2, and -3, are constitutively expressed
in the brain. We examined the regulatory mechanisms governing the
expression level of Vesl-1S protein, either an exogenously introduced
one in COS7 or human embryonic kidney 293T cells or an
endogenous one in rat brain neurons in cultures. In both cases,
application of proteasome inhibitors increased the amount of Vesl-1S
protein but not that of Vesl-1L, -2, or -3 protein. Deletion analyses
revealed that the C-terminal 11-amino acid region was responsible for
the proteolysis of Vesl-1S by proteasomes. Application of proteasome
inhibitors promoted ubiquitination of Vesl-1S protein but not that of
the Vesl-1S deletion mutant, which evaded proteasome-mediated
degradation. These results indicate that ubiquitin-proteasome
systems are involved in the regulation of the expression level of
Vesl-1S protein.
Long term potentiation
(LTP),1 which is thought to
underlie mechanisms of learning and memory, has two distinct phases.
The early-phase LTP lasts for no more than several hours and does not
depend on protein synthesis, whereas the late-phase LTP (L-LTP) lasts
for weeks in vivo and depends on de novo RNA
transcription and protein synthesis (1-3). The formation of long term
memory requires de novo RNA transcription and protein
synthesis (4-6). Thus, activity-dependent gene expression
is expected to play a critical role in long term memory.
Vesl-1S/Homer-1a was isolated as a gene whose expression was
up-regulated following LTP induction (7, 8). Vesl-1L/Homer-1c/PSD-Zip45 and Vesl-1L( The level of vesl-1S mRNA in the hippocampus is
drastically increased during seizure and LTP, but the increase in the
amount of Vesl-1S protein is limited (9). Moreover, all members of Vesl
family contain PEST sequences that are thought to be
ubiquitin-proteasome-dependent degradation signals (15). We
considered that the amount of Vesl-1S protein might be regulated by
certain proteases. The ubiquitin-proteasome pathway, one of the protein
degradation systems of the cell, is involved in a variety of cellular
processes, for instance, cell cycling (16), transcriptional activation
(17), apoptosis (18), circadian rhythm (19), neurodegeneration (20),
and neuronal plasticity (21, 22). Protein ubiquitination involves three classes of enzymes, E1 ubiquitin-activating enzymes, E2
ubiquitin-conjugating enzymes, and E3 ubiquitin-protein ligases. In
these multienzyme pathways, target proteins are conjugated with
polymers of ubiquitin, which trigger their rapid degradation by
proteasomes (23). As the expression level of vesl-1S
mRNA does not readily parallel that of Vesl-1S protein after L-LTP
induction, we investigated the effects of protease inhibitors on the
amount of Vesl-1S protein. We found that proteasome inhibitors promoted
the expression and ubiquitination of Vesl-1S proteins and identified a
proteolytic signal sequence that controlled its ubiquitinations.
Chemicals--
E-64-d
(2S,3S-t-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl
ester) and MG132
(carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) were purchased form Peptide Institute Inc. (Osaka, Japan). Lactacystin was purchased from Calbiochem. These compounds were dissolved in
Me2SO before use, and throughout the experiments,
the final concentration of Me2SO in cell culture
medium, including control culture medium, was kept at 0.1%.
Cell Culture--
For Western blot analyses, cortical cells were
used. Rat neurons were cultured as follows. Brains of embryonic Wistar
rats (E18-19) were rapidly removed, dissected, and incubated at
37 °C for 10 min in papain solution containing the following: 5 mM L-cysteine, 1 mM EDTA, 10 mM HEPES-NaOH (pH 7.3), 100 µg/ml bovine serum albumin,
10 units/ml papain (Worthington), and 0.02% DNase (Sigma). The
reaction was stopped by adding an equal volume of heat-inactivated
horse serum (Life Technologies, Inc., Grand Island, NY). Cells were
filtered through lens paper, plated onto polyethyleneimine-coated 60-mm
dishes at 8 × 106-4 × 106
cells/dish, and cultured in Dulbecco's modified Eagle's medium supplemented with 100 units of penicillin G/ml, 10 µg of streptomycin sulfate/ml, 4 mM glutamine, and 10% horse serum.
The COS7 cells and the HEK293T cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 100 units of penicillin G/ml, 10 µg of streptomycin sulfate/ml, 4 mM glutamine,
and 10% (v/v) fetal bovine serum. Cultures were maintained at 37 °C
in a humidified atmosphere containing 5% CO2.
Construction of FLAG-tagged Proteins and HA-tagged
Ubiquitin--
FLAG (DYKDDDK)-tagged Vesl constructs and Vesl-1S
deletion mutants were generated by polymerase chain reaction using
specific primers and subcloned into the mammalian expression vector
pcDNA3 (Invitrogen, Carlsbad, CA), which contains the neomycin
resistance gene (NPT II). FLAG tags were inserted between the
initiation codon and the codon for the second amino acid to construct
FLAG-Vesl-1L, -1S, -2, and -3. FLAG-tagged I
N-terminal HA-tagged ubiquitin was generated by the
polymerase chain reaction using high fidelity thermostable DNA
polymerase KOD (Toyobo, Tokyo, Japan) with the following
primers:
5'-ATAGATATCGCCACCATGGCCTACCCATACGACGTCCCAGACTACGCTCAGATCTTCGTGAAAACCCTTACC-3' and 5'-ATAGATACTTTAACCACCTCTCAGACGCAGGAC-3'. The polymerase chain reaction product was digested with EcoRV, subcloned
into pBluescript II SK+, and sequenced. pBluescript II SK+ was digested
with EcoRI and XhoI and subcloned into pcDNA3.
COS7 Cell Transfections and Immunoblots--
Transfections
of DNA constructs into COS7 cells were performed with 50 µg of each
plasmid DNA by electroporation (Electro Cell Manipulator 600;
BTX) according to the manufacturer's instructions. 48 h
after transfection, cells were treated with drugs for 10 h and
then cells were extracted in 2× SDS sample buffer. Equal amounts of
cell extract were separated by SDS polyacrylamide gel electrophoresis
(12.5% polyacrylamide). After transfer of the separated proteins to a
polyvinylidene difluoride membrane, the membrane was fixed for 45 min
with 4% paraformaldehyde in phosphate-buffered saline at 4 °C and
rinsed three times for 20 min with phosphate-buffered saline.
FLAG-tagged Vesl proteins from transfected COS7 cells were detected by
using a monoclonal antibody (anti-FLAG M5 antibody; Eastman Kodak Co.)
and the Vistra ECF Western blotting system (Amersham Pharmacia
Biotech). Vesl-1S proteins from cultured neuron extracts were detected
with rabbit antibody raised against recombinant Vesl-1S protein and
visualized by horseradish peroxidase-conjugated anti-rabbit antibody
(Amersham Pharmacia Biotech) and a SuperSignal West Femto maximum
sensitivity substrate (Pierce). NPT II proteins were detected with
polyclonal antibody (rabbit anti-neomycin polyclonal antibody; 5 Prime
Immunoprecipitation--
HEK293T cells were transfected with DNA
constructs by the calcium phosphate method. After 48 h, the cells
were lysed with a solution containing 50 mM Tris-Cl (pH
7.6), 300 mM NaCl, 0.5% Triton X-100 (v/v), aprotinin (10 µg/ml), leupeptin (10 µg/ml), 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 0.4 mM Na3VO4, 0.4 mM EDTA, 10 mM NaF, and 10 mM sodium pyrophosphate. The
cell lysates were pretreated with 30 µg of protein A/G-Sepharose beads (Santa Cruz Biotechnology) for 30 min at 4 °C and were then incubated with 5 µg of FLAG antibody (M2; Sigma) and protein
A/G-Sepharose beads for 4 h at 4 °C. The resulting
immunoprecipitates were then washed thoroughly four times with ice-cold
lysis buffer and subjected to immunoblot analyses with antibodies to
FLAG or to ubiquitin (1B3; MBL, Nagoya, Japan).
The Amount of Vesl-1S Protein, but Not That of Vesl-1L, -2, or -3, Is Increased by Proteasome Inhibitors--
To investigate the turnover
of proteins of the Vesl family, we constructed plasmids that expressed
Vesl proteins containing FLAG tags (Fig.
1A). These constructs did not
contain untranslated regions. The expression of these mRNAs was
regulated by the same promoter, and these Vesl proteins were translated
by the same initiation signal. The plasmids were introduced into COS7
cells. The effects of protease inhibitors on the levels of Vesl
proteins were evaluated by immunoblotting using anti-FLAG antibody. We used specific inhibitors for proteasomes and lysosomal proteases. Two
types of proteasome inhibitors, MG132 and lactacystin, which are
structurally unrelated, significantly increased the amount of Vesl-1S
protein, whereas the amounts of Vesl-1L, -2, and -3 were not affected.
An inhibitor of proteases of the lysosomes and calpain family, E-64-d,
had no effect on the amount of any protein of the Vesl family (Fig. 1,
B and C). These results indicate that Vesl-1S,
but not Vesl-1L, -2, or -3, undergoes rapid degradation by
proteasomes.
The C-terminal 11-Amino Acid Region of Vesl-1S Is Responsible for
Its Degradation by Proteasomes--
The next step in our study was to
identify the region of the Vesl-1S protein responsible for the
regulation of its degradation by proteasomes. We introduced deletion
constructs of FLAG-tagged Vesl-1S cDNA into COS7 cells, and
the levels of the three truncated Vesl-1S proteins, VSD-1, -2, and -3 (Figs. 2A), were
evaluated by immunoblotting using anti-FLAG antibody. The unevenness in the transfection efficiencies of vectors carrying VSD-1, -2, and -3 was
normalized to the level of the NPT II protein expressed from the
same vector. As Fig. 2, B and C shows, the
expression levels of VSD-1, -2, and -3 were remarkably higher than that
of Vesl-1S protein. These results indicate that the 11-amino
acid region in the C terminus of Vesl-1S reduces the stability of the Vesl-1S protein.
To investigate the mechanism responsible for the increase in the
amounts of VSD-1 and -3, we examined the effects of the protease inhibitors MG132, lactacystin, and E-64-d on the levels of these truncated proteins (Fig. 3, A
and B). No effect was observed, indicating that the increase
in the level of truncated Vesl-1S protein was because of its resistance
to proteolysis by proteasomes. As the 11-amino acid region is unique to
Vesl-1S among Vesl family proteins, this sequence most likely
predestines Vesl-1S for rapid degradation.
The Amount of Endogenous Vesl-1S Protein Is Increased by
Application of a Proteasome Inhibitor to Cultured Neurons--
We
investigated whether the turnover of endogenous Vesl-1S protein was
also affected by the application of proteasome inhibitors to cultured
neurons. The amount of Vesl-1S protein was measured by Western blot
analysis using the anti-pan-Vesl antibody, which recognizes all members
of the Vesl family of proteins. As the molecular mass of Vesl-1S
protein is significantly lower than that of other members of the Vesl
family, we could identify Vesl-1S immunosignals after SDS
polyacrylamide gel electrophoresis. The mobility of endogenous Vesl-1S
protein during SDS polyacrylamide gel electrophoresis was confirmed by
loading bacterially expressed Vesl-1S side by side on the
polyacrylamide gel. After the application of MG132, we observed strong
immunosignals at 28 kDa, which is a slightly lower molecular mass than
that of the recombinant proteins. No immunosignals at the corresponding
position were detected under control conditions. These Vesl-1S
immunoreactivities were blocked by incubation of the anti-Vesl antibody
with the GST-Vesl-1S protein (Fig. 4).
MG132 did not affect the intensity of immunosignals at 48 kDa, which
corresponded to those of Vesl-1L, -2, and -3. These results indicate
that only endogenous Vesl-1S among Vesl family members is selectively
degraded by proteasomes in cultured neurons and strongly suggest that
the level of Vesl-1S protein is regulated by the proteasome pathway in
neurons.
Ubiquitination of Vesl-1S Protein--
We next investigated
whether the degradation of Vesl-1S protein was regulated by ubiquitin
signals. To investigate its ubiquitination, we co-expressed FLAG-tagged
Vesl-1S or Vesl-1S mutants with HA-tagged ubiquitin in HEK293T cells
and examined their interaction by immunoprecipitation with anti-FLAG
antibody. We found that the treatment of cells with the proteasome
inhibitor (MG132) not only led to the accumulation of Vesl-1S protein
but also promoted the accumulation of multiubiquitinated Vesl-1S. In
contrast, the deletion mutant VSD-3, which was not degraded by
proteasomes, was not altered to multiubiquitinated forms by the
treatment of the proteasome inhibitor (Fig.
5A).
It is known that ubiquitin is attached to the lysine residues in target
proteins (25). The C-terminal Vesl-1S-specific region, which is
responsible for proteolysis of Vesl-1S by proteasomes, contains one
lysine residue. To examine whether this lysine residue is essential for
ubiquitination of Vesl-1S, we constructed a Vesl-1S mutant in which
this lysine residue was replaced with an arginine residue (V1S-K186R;
see Fig. 5B). We found that the ubiquitination and the
expression of the V1S-K186R were both promoted by the proteasome
inhibitor. There was little difference in the extent of ubiquitination
between the mutant and the wild type (Fig. 5, C and
D). These results suggest that the site of ubiquitination of
Vesl-1S may most likely be lysine residues other than this lysine residue.
All Vesl family proteins contain PEST sequences (Fig.
1A), which are thought to act as proteolytic signals (15).
We have reported here that Vesl-1S, but not Vesl-1L, -2, or -3, is
degraded by proteasomes and that the 11-amino acid region in the C
terminus of Vesl-1S, which is the unique sequence among Vesl family
proteins, is responsible for proteasome-mediated proteolysis. As the
stable deletion mutants contained PEST sequences, it seems likely that PEST sequences are not involved in the proteasome-mediated degradation of Vesl-1S protein, although we will need to examine the stability of a
deletion mutant lacking only the PEST sequences to verify this hypothesis.
The signals for protein degradation by proteasomes are often
ubiquitination. In the case of Vesl-1S, we also found that it was
heavily ubiquitinated. In contrast, the proteasome-resistant stable
mutant of Vesl-1S, VSD-3, was not ubiquitinated. Therefore the
ubiquitination signals may most likely reside in the C-terminal 11-amino acid region, and the ubiquitination of Vesl-1S may promote the
rapid degradation of the protein by proteasomes. At present, we have
not identified the sites of ubiquitination, but at least the C-terminal
11-amino acid region does not seem to contain the only or major
ubiquitination site. Thus, this region may function as a signal to
stimulate ubiquitination of the other sites of Vesl-1S protein.
The level of endogenous Vesl-1S protein in neurons is also regulated,
at least partly, by proteasomes. The level of vesl-1S mRNA is increased after L-LTP induction, although the expression of
vesl-1L mRNA is not modulated during L-LTP. After L-LTP
induction, the level of vesl-1S mRNA is higher than that
of vesl-1L mRNA, although the amount of Vesl-1S protein
is lower than that of Vesl-1L protein (9). Following L-LTP induction,
Vesl-1S proteins accumulated in the portion of the dendrites that had
undergone synaptic activation (26). The mRNA for Arc, which was
isolated as a synaptic plasticity-regulated gene, is localized to the
active postsynaptic regions of dendrites (27) (28). In contrast,
vesl-1S mRNA remains in the cell body after L-LTP
induction (8). It is likely that newly synthesized Vesl-1S proteins are
rapidly degraded by proteasomes following L-LTP induction and that the
overall amount of Vesl-1S protein is relatively low. However, when
Vesl-1S proteins evade proteasome-mediated degradation by some unknown
mechanism, these proteins may accumulate in postsynaptic regions.
An unresolved issue is how proteasome-mediated degradation of Vesl-1S
proteins is prevented. What is the inhibition signal? The stability of
proteins degraded by proteasomes is regulated by phosphorylation in
many cases. Recently, we found that phorbol esters (phorbol
12-myristate 13-acetate or phorbol 12,13-dibutyrate) promoted the
punctate distribution of Vesl-1S in neurons and that these phenomena
were observed in the absence of de novo protein synthesis.2 Phorbol esters
activate several types of proteins (29). As some proteins (Mos and p53)
evade proteasome-mediated degradation when phosphorylated (30-33), it
is possible that the application of phorbol esters may provoke the same
phenomena as those induced by proteasome inhibitors in neurons. The
Vesl-1S protein may be modified by some kind of kinases activated by
phorbol esters and, thereby, evade degradation by proteasomes. A
possible hypothesis is that Vesl-1S protein accumulates selectively in
certain synapses when proteasomes are unable to degrade the modified
Vesl-1S protein present at these synapses. The accumulation of Vesl-1S
protein at such synapses might affect the cell surface expression of
group I metabotropic glutamate receptors 1/5 and promote remodeling of
synapses, considering the recent observations that the cell surface
expression of group I metabotropic glutamate receptors 1/5 were
increased when co-expressed with Vesl-1S and that this increase was
inhibited by Vesl-1L (34, 35).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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12)/Homer-1b, which are splice variants of Vesl-1S, are
constitutively expressed in the brain. Vesl-2/Homer-2b,
Vesl-2(
11)/Homer-2a, and Vesl-3/Homer-3 are highly related to
Vesl-1L in that both contain EVH1 domains in their N termini and
leucine zippers in their C termini that mediate multimerization
(9-11). The EVH1 domains of Vesl family proteins interact with group I
metabotropic glutamate receptors 1/5 (7) and inositol trisphosphate
receptors (12). Moreover, Vesl family proteins interact with the Shank protein, which binds to the NMDAR·PSD-95·GKAP complex and
cortactin (13, 14). Thus Vesl family proteins may be a component of huge PSD-95 protein complexes located in postsynaptic regions.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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B was prepared as
described (24).
3 Prime, Inc., Boulder, CO).
RESULTS
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Fig. 1.
Vesl-1S, but not Vesl-1L, -2, or -3, is
degraded by proteasomes. A, schematic structures of
members of the Vesl family of proteins. Closed boxes
indicate FLAG tags. Hatched boxes indicate PEST sequences.
Vesl-1S and Vesl-1L have two PEST sequences (PEST scores, 11.53 and
6.60). Vesl-2 and -3 have one PEST sequence each (PEST scores, 14.23 and 5.64, respectively). These PEST sequences were found using PEST
find programs. B, Western blot analyses of Vesl
proteins from COS7 cells. COS7 cells were transfected with FLAG-tagged
vesl family cDNA, and these cells were cultured with
0.1% Me2SO, 10 µM MG132, 10 µM
lactacystin, or 10 µM E-64-d for 10 h. The Vesl
proteins in the extracts were detected with FLAG antibody.
C, quantitative analyses of the data depicted in
B. Quantities are indicated relative to the levels of Vesl
proteins observed after Me2SO treatment. Error
bars show S.E.
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Fig. 2.
The stability of Vesl-1S protein is increased
by the deletion of its C-terminal region. A, schematic
structures of Vesl-1S deletion mutants. B, Western blot
analyses of Vesl-1S deletion mutants from transfected COS7 cells. The
top panels indicate immunoblots of Vesl-1S mutants using
FLAG antibody, and the bottom panels indicate NPT II
immunoblots, which served as internal controls. The NPT II gene was
carried by all the expression vectors. C, quantitative
analyses of the data depicted in B. Quantities are indicted
relative to the amount of wild-type Vesl-1S protein. Error
bars show S.E.
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Fig. 3.
The proteasome inhibitor MG132 had no effect
on the amount of truncated Vesl-1S protein lacking the
C-terminal-specific region. A, Western blot analyses of
Vesl-1S deletion mutants from transfected COS7 cells, which were
cultured with 0.1% Me2SO, 10 µM MG132, 10 µM lactacystin, or 10 µM E-64-d for 10 h. The cell extracts were blotted with FLAG antibody. B,
quantitative analyses of the data depicted in A. Quantities
are indicated relative to the amounts of VSD proteins observed after
Me2SO treatment. Error bars show S.E.
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Fig. 4.
The amount of Vesl-1S protein is increased by
the application of proteasome inhibitors to cultured neurons.
Extracts of dissociated cultures of cortices (21-28 days in
vitro) and purified recombinant Vesl-1S protein were
Western-blotted with Vesl antibody (lanes 1, 2,
and 3) or Vesl antibody preabsorbed with GST-Vesl-1S fusion
protein (lanes 4 and 5). Lane 1, cells
cultured with 0.1% Me2SO for 10 h. Lanes 2 and 4, cells cultured with 10 µM MG132 for
10 h. Lanes 3 and 5, bacterially expressed
Vesl-1S containing six additional amino acids, which were
generated by cleaving the GST-Vesl-1S fusion protein with
protease Factor-Xa.
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Fig. 5.
Vesl-1S and V1S-K186R but not VSD-3 are
degraded by the ubiquitin-proteasome pathway. A,
immunoprecipitation analyses of Vesl-1S proteins from HEK293T cells.
HEK293T cells were transfected with FLAG-tagged Vesl-1S, VSD-3, or
I Ba (positive control), in combination with HA-tagged ubiquitin, and
these cells were cultured with 0.1% Me2SO or 10 µM MG132 for 10 h. Cell lysates were subjected to
immunoprecipitation (IP) with FLAG antibody, and the
resulting precipitates were subjected to immunoblot (IB)
analyses with antibodies to ubiquitin or FLAG. The high molecular mass
ubiquitinated proteins are shown as (Ub)n on the
right. The I
B, which is associated with NF-
B,
was used as a positive control for ubiquitination. Under normal
conditions, only a small fraction of I
B is ubiquitinated and rapidly
degraded by proteasomes, but the remaining fraction, which is not
ubiquitinated, is stable (36). As anti-ubiquitin immunoblot is more
sensitive than anti-FLAG immunoblot, we could detect the difference of
ubiquitination. In contrast to I
B, Vesl-1S is much less stable and
is mostly degraded in the absence of the proteasome inhibitor. Thus
Vesl-1S could be detected weakly, whether ubiquitinated or not, in the
absence of the inhibitor. B, alignment of C-terminal amino
acid sequences of Vesl-1S and its mutants. Bold characters
represent the Vesl-1S-specific region. C,
immunoprecipitation analyses of the Vesl-1S mutant protein, V1S-K186R,
with a Lys to Arg mutation in the C-terminal Vesl-1S-specific region.
D, quantification of ubiquitination of Vesl-1S and V1S-K186R
depicted in C. The band intensity of multiubiquitinated
V1S-K186R normalized by the intensity of non-ubiquitinated V1S-K186R is
shown relative to that of Vesl-1S. The error bar shows
S.E.
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FOOTNOTES |
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* 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. Tel.: 81-92-642-2630; Fax: 81-92-642-2645; E-mail: hsugiscb@mbox.nc.kyushu-u.ac.jp.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M011097200
2 Kato, A., Fukuda, T., Fukazawa, Y., Isojima, Y., Fujitani, K., Inokuchi, K., and Sugiyama, H. (2001) Eur. J. Neurosci. 7, 1292-1302.
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ABBREVIATIONS |
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The abbreviations used are: LTP, long term potentiation; L-LTP, late-phase LTP; HEK, human embryonic kidney; HA, hemagglutinin; NPT, neomycin phosphotransferase; GST, glutathione S-transferase..
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