From the Center for Cardiovascular Research,
University of Rochester School of Medicine and Dentistry, Rochester,
New York 14642, the
Department of Pathology, University of
Washington School of Medicine, Seattle, Washington 98195, and the
** Department of Immunology, Scripps Research Institute, La Jolla,
California 92037
Received for publication, October 11, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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The mitogen-activated protein kinases
(MAPKs) play important roles in regulation of cell growth and survival.
Human MAPK 5 (ERK5) or Big MAP kinase 1 (BMK1) is a recently
cloned member of the MAPK family. To identify ERK5-related kinases, we
searched the GenBankTM expressed sequence tag (EST) data base for
mouse cDNAs with homology to human ERK5. A full-length mouse
cDNA that was highly homologous to the human ERK5 was identified.
Further analysis of ERK5 polymerase chain reaction products generated from mouse embryo cDNA yielded three mouse ERK5 cDNAs (mERK5a, mERK5b, and mERK5c). Sequence analysis showed that these cDNAs are
alternative splice products of the mouse ERK5 gene.
Interestingly, expressed mERK5b and mERK5c act as dominant negative
inhibitors based on inhibition of mERK5a kinase activity and
mERK5a-mediated MEF2C transactivation. However, the physiological
significance of mERK5b and mERK5c is not fully understood. Further
investigation using these mouse ERK5 splice variants and other
constructed mutants identified functional roles of several regions of
mERK5, which appear to be important for protein-protein interaction and
intracellular localization. Specifically, we found that the long
C-terminal tail, which contains a putative nuclear localization signal,
is not required for activation and kinase activity but is responsible for the activation of nuclear transcription factor MEF2C due to nuclear
targeting. In addition, the N-terminal domain spanning amino acids
(aa) 1-77 is important for cytoplasmic targeting; the domain
from aa 78 to 139 is required for association with the upstream kinase
MEK5; and the domain from aa 140-406 is necessary for
oligomerization. Taken together, these observations indicate that ERK5 is regulated by distinct mechanisms determined by its unique
structure and presumably the presence of multiple splice variants.
Mitogen-activated protein kinases
(MAPKs)1 are serine/threonine
protein kinases that play important roles in signal transduction pathways activated by extracellular stimuli. MAPKs regulate many cellular processes, including cell proliferation, cell differentiation, cell death, and stress responses (1, 2). MAPKs constitute a superfamily
of highly related serine/threonine kinases. At least seven family
members of the MAPK family have been identified in mammals: ERK1/2
(extracellular signal-regulated kinase 1 and 2) (3, 4), JNK/SAPK (c-Jun
N-terminal kinase/stress-activated protein kinase) (5-7), p38 (a
mammalian equivalent of the yeast high-osmolarity glycerol kinase) (6),
Big MAP kinase 1 (BMK1, also known as ERK5) (8, 9), ERK6
(mitogen-activated protein kinase 6) (10), and ERK7 (extracellular
signal-regulated kinase 7) (11). MAPKs are activated by phosphorylation
on threonine and tyrosine residues in a Thr-X-Tyr
(TXY) motif involving upstream dual-specificity
protein kinases (MAPK kinases) and phosphatases. The TXY
activation motif is used to classify the MAPK superfamily into three
main groups. The TEY (Thr-Glu-Tyr) group consists of extracellular
signal-regulated kinases ERK1/2, ERK5, and ERK7. The TPY (Thr-Pro-Tyr)
family consists of JNK/SAPK (5). The TGY (Thr-Gly-Tyr) family includes
p38 and ERK6. Each MAPK pathway generally consists of three kinase
modules composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase
(MAPKKK) (12). These kinase modules are differentially activated by a
variety of cellular stimuli and contribute to distinct cellular
function (13). The ERK1/2 module includes Raf isoforms, MEK1/2 and
ERK1/2, which are highly responsive to mitogenic signals such as growth
factors and cytokines. In contrast, JNK/SAPK and p38 are
stress-sensitive pathways activated by MEK 4/7 and MEK3/6,
respectively. ERK5 is specifically activated by MEK5 (14).
Human ERK5 was recently cloned by two groups (8, 9). Human ERK5
contains 816 amino acid residues with a primary structure distinct from
other MAPK members. ERK5 has a unique long C-tail and a distinct
loop-12 domain. ERK5 is activated by reactive oxygen species (15),
hyperosmolarity (16), and fluid shear stress (16). Most recently, it
has been shown that ERK5 is required for EGF-induced cell proliferation
and progression through the cell cycle (17). Although ERK5 has a TEY
motif in its dual phosphorylation site similar to ERK1/2 and ERK7,
several studies have shown that ERK5 has different upstream activators
and downstream substrates compared with other MAPKs.
In the present study, we report the identification of three
alternatively spliced mouse ERK5 cDNAs, termed mERK5a, mERK5b, and
mERK5c. The putative protein sequences deduced from these three
cDNAs are identical except in their N-terminal regions. mERK5b and
mERK5c lack N-terminal 69 amino acids and 139 amino acids,
respectively, compared with mERK5a. It is likely that the variations in
N-terminal sequences are produced by alternatively choosing different
splicing donors and acceptors within a single intron. More
interestingly, mERK5b and mERK5c, which are catalytically inactive, act
as dominant negative kinases. Finally, by using N- or C-terminal
truncated mERK5, we found that three N-terminal domains spanning aa
1-77, aa 78-139, and aa 140-406 are important for cytoplasmic
targeting, association with the upstream kinase MEK5, and
oligomerization, respectively. The C-terminal tail is essential for the
biological activity of ERK5 in vivo by mediating nucleus
translocation, which is dependent upon the NLS in the C-terminal
region. These observations should help us understand the biological
reasons for this diversity in ERK5 and may provide new approaches to
modify ERK5 signaling.
Isolation of Mouse ERK5 Isoforms--
The GenBankTM expressed
sequence tag (EST) data base was searched using the program BLAST with
amino acid sequences corresponding to the human ERK5. Several EST
fragments displayed high degrees of amino acid homology. Among them, a
full-length mouse clone was obtained from Genome Systems Inc
(GenBankTM accession number AA288345). A manual sequencing method with
the Sequetherm Cycle sequencing kit (Epicentre Technologies Corp.) was
used to sequence this clone according to the manufacturer's protocols.
Several PCR fragments were found in the first-strand cDNAs of a
mouse embryo (purchased from CLONTECH) and were
subcloned into the TA vector (Invitrogen) for sequencing. Combining
analysis of mouse ERK5 RT-PCR clones with mouse EST clones, three mouse
ERK5 cDNAs were identified. To determine the mechanism for
generating three mouse ERK5 isoforms, a mouse genomic DNA fragment
encompassing the mouse ERK5 isoforms splicing junction was isolated by
PCR amplification using oligonucleotides 5'-ACGAGTACGAGATCATCGAGACC-3' and 5'-GGTCACCACATCAAAAGCATTAGG-3'.
Construction of Expression Plasmids--
Clones for mouse ERK5a
(nucleotides 10-2776), ERK5b (nucleotides 280-2826), and ERK5c
(nucleotides 837-3196) were subcloned into the multiple cloning
regions of the pcDNA3.1/His vectors (Invitrogen). All isoforms were
fused in-frame with an N-terminal Xpress tag. mERK5a(-tail) (aa 1-406)
was constructed by removing aa 407-806 with PstI digestion.
GFP-tail and GFP-NLS were generated by ligation of the PCR-amplified
C-tail (aa 407-806) and NLS fragment (aa 505-539), respectively,
containing the artificial restriction sites BamHI and
EcoRI to the pEGFP-C3 (CLONTECH) cut
with BamHI and EcoRI. mERK5a(-NLS), lacking aa
505-539, was generated by PCR with the Expand High-Fidelity PCR system
(Roche Molecular Biochemicals). Briefly, an antisense primer matching
the DNA sequence upstream of the codon aa 505 and a sense primer
downstream of the codon aa 539 were generated. PCR reaction was
performed using the entire mERK5a in the PcDNA3.1/His vector as the
template, and the PCR product was treated with T4 DNA polymerase to
create a blunt end. Purified PCR product was further treated with
polynucleotide kinase and T4 ligase and subsequently transformed into
the competent bacterial DH5 Cell Culture and Transient Expression--
CHO-K1 were
maintained in DMEM/F-12 medium (Life Technologies, Inc) supplemented
with 10% calf serum, 50 units/ml penicillin and 50 µg/ml
streptomycin. Cells were transiently transfected at 50-80% confluence
using LipofectAMINE reagent (Life Technologies, Inc.) and harvested
48 h after transfection.
Western Blot Analysis--
Cells were washed with
phosphate-buffered saline (PBS) and 0.2 ml of TME lysis buffer (10 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 25 mM NaF) containing fresh 100 µM Na3VO4, 20 µg/ml leupeptin, 1 µg/ml pepstatin A, 4 µg/ml aprotinin, and 1 mM
dithiothreitol (18). Cell lysates were prepared by scraping,
sonication, and centrifugation.
Mouse embryos (15 day) were homogenized in 2.0 ml of lysis buffer (50 mM sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 µM Na3VO4, 10 mM
HEPES, pH 7.4, 0.1% Triton X-100, 500 µM
phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin).
Cell fractionation was performed by extraction with different buffer
and sequential centrifugation. First, cells were lysed in the TME lysis
buffer contain 0.1% Triton X-100, and the cell lysate was centrifuged
at 10,000 × g for 1 h. The supernatant was
collected as the cytoplasmic fraction. The pellet was then resuspended
in the lysis buffer containing 1% Triton X-100 and centrifuged at
10,000 × g for 1 h. The supernatant was collected as the nuclear fraction. The pellet was further resuspended in the RIPA
buffer (20 mM Tris-HCl, pH 7.5, 2.5 mM EDTA,
1% Triton X-100, 10% glycerol, 1% deoxycholic acid, 0.1%
SDS, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and centrifuged at
10,000 × g for 1 h. The supernatant was collected
as the cytoskeleton fraction, and the pellet was resuspended in sample
buffer as the nuclear fraction.
Cell lysates, cellular fractionates, or tissue extracts were boiled in
the presence of 1× sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 4% Immunoprecipitation and Immune Complex Kinase Assay--
Immune
complex kinase assays were performed with ectopically expressed tagged
ERK5 proteins from CHO-K1 cells as previously described (19), except 2 µg of MBP was used per reaction in kinase buffer. Proteins were
separated by 15% SDS-PAGE, transferred to a nitrocellulose, and
subjected to autoradiography. The presence of epitope-tagged proteins
in immunoprecipitates was verified by Western analysis with antibody
against the tag.
RNA Isolation and RT-PCR--
Total RNA was prepared from
multiple adult mouse tissues and mouse embryos (15 days) using an
RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.
Two degenerate oligonucleotide primers were designed corresponding to
splicing junction sequences of three cDNAs: the sense primer
5'-ACGAGTACGAGATCATCGAGACC-3' and antisense primer
5'-GGTCACCACATCAAAAGCATTAGG-3'. The first-strand cDNA was
synthesized by Superscript II reverse-transcriptase (Life Technologies,
Inc.) with antisense primer. The amplification was carried out in a
100-µl mixture containing 2 µl of the first-strand cDNA
product, 10 µM each of the sense and antisense primer,
and 5 units of Taq DNA polymerase (Life Technologies, Inc.).
The PCR reaction was performed as follows: initial denaturation at
94 °C for 3 min and 30 cycles of amplification (denaturation at
94 °C for 1 min, annealing at 55 °C for 2 min, extension at
72 °C for 2 min), followed by a final extension step of 10 min at
72 °C. Reactions were electrophoresed on a 1.5% agarose gel.
Reporter Gene Expression--
The MEF2C fusion activator vector,
encoding the GAL4-binding domain fused to MEF2C activation domain (14),
was cotransfected into CHO-K1 cells along with the GAL4-responsive
reporter plasmid pG5E1bLUC, which contains five GAL4 sites cloned
upstream of a minimal promoter driving a luciferase gene (14).
For transfection, CHO-K1 cells (0.2 × 106 cells per
well) were seeded into 24-well plates the day before transfection.
Cells were transfected with 0.5 µg of DNA in total per well using
LipofectAMINE Plus (Life Technologies). After 5 h, the
transfection was stopped by adding equal volume of DMEM/F-12 (10%
fetal bovine serum). After 24 h, the medium was changed to a
serum-free DMEM/F-12 for an additional 24 h. Then cells were collected for luciferase assay. In the cases of testing the endogenous ERK5, 10% serum was added and the cells were incubated for an additional 4 h before harvesting. A green fluorescence protein (GFP) expression vector (pEGFP-N1, from CLONTECH)
was used to control for transfection efficiency. The total amount of
DNA for each well was kept constant using the empty vector
pcDNA3.1/His (Invitrogen). Luciferase assays were performed with a
Luciferase Reporter Gene Assay kit (Roche Molecular Biochemicals) as
instructed. Briefly, cells were washed twice with PBS and lysed in 200 µl of lysis buffer at room temperature for 15 min with shaking. 50 µl of cell extracts was transferred into a 96-well microtiter plate.
The fluorescence intensity of GFP was measured using a Wallace
multicounter (Wallace). 50 µl of luciferase substrate were then added
to the cell lysates, and the luciferase activities were determined by
measuring luminescence intensity using the same Wallace multicounter.
To correct for transfection efficiency, the luciferase activity was
divided by the green fluorescence intensity.
Immunocytochemistry--
CHO-K1 cells, grown on LabTek II
chamber slides, were cotransfected with Xpress-tagged ERK constructs in
the presence of either pCDNA3 vector or MEK5(D). Following the
transfection for 48 h, the cells were then fixed in 10% formalin
in PBS for 15 min, washed, blocked, and incubated with anti-Xpress
antibody at a 1:2000 dilution. After a 1-h incubation at room
temperature, the cells were washed and then incubated with
fluorescein-conjugated anti-mouse antibody (Vector) at a 1:200
dilution. The stained cells were analyzed under an Olympus Fluoview
confocal microscope.
Cloning of Three Different Mouse ERK5 Splice Variants and Genomic
Sequence Analysis--
To identify mouse ERK5 cDNAs, the EST data
base was searched, and several EST clones with high homology to human
ERK5 were found. Among these, a full-length EST cDNA clone
(AA288345) was purchased and sequenced. Comparing this sequence with
the published human ERK5 sequence (8, 9), we found that this cDNA
clone contained an insert as well as an in-frame stop codon within the
insert. This suggested that multiple ERK5 cDNAs might exist.
To confirm the presence of diverse species of ERK5 cDNAs in the
mouse, mouse embryo first-strand cDNA (from
CLONTECH) was used for PCR with primers from
conserved regions of human and mouse ERK5. Sequence analysis showed
that at least three different ERK5 cDNAs were present, which we
will designate as mERK5a, mERK5b, and mERK5c (Fig.
1A). mERK5a is the mouse
cDNA most homologous to human ERK5. mERK5b and mERK5c cDNAs
contain one or two inserts compared with mERK5a, respectively. Although
mERK5b has a 50-bp insert (I-1) (Fig. 1A),
mERK5c, which corresponds to the mouse EST clone mentioned above,
contains both the 50-bp (I-1) insert found in mERK5b and a
91-bp (I-2) insert (Fig. 1A). Both inserts start
with consensus splice donor "gt," and the second insert (I-2) ends with an acceptor sequence "ag," suggesting
that they are likely to be intron sequences that were not spliced
out.
To explore the mechanism for producing these three different mouse ERK5
cDNAs, PCR and sequencing of the mouse genomic DNA flanking the
splicing region of the ERK5 gene was performed. The genomic
structure of the splicing junction of mouse ERK5 gene is
shown in Fig. 1B. Sequence analysis of genomic DNA showed an additional 429-bp (I-3) insert with consensus "gt/ag"
just after I-2. It is likely that the variations in
N-terminal sequences are produced by alternatively choosing different
splicing donors and acceptors within a single intron that is composed
of I-1, I-2, and I-3. mERK5a is likely
generated by use of the splicing donor D1 and the acceptor A2, whereas
mERK5b is generated by use of D2 and A2 and mERK5c is generated by use
of D3 and A2. The I-1 intron introduces a stop codon and
causes mERK5b and mERK5c to have shorter N termini than mERK5a.
Protein Sequence Analysis--
mERK5a contains a putative open
reading frame (ORF) from nucleotide 27 to nucleotide 2447 that encodes
a protein of 806 amino acids with a predicted molecular mass of 88 kDa,
whereas mERK5b (putative ORF: nucleotides 284-2497) contains 737 amino
acid with a predicted molecular mass of 80 kDa and mERK5c (putative
ORF: nucleotide 864-2867) consists of 667 amino acid residues with a
predicted molecular mass of 73 kDa. The predicted N-terminal amino acid
sequences for the three isoforms of ERK5 are shown in Fig.
2A. It is important to note
that mERK5b and mERK5c lack the GXGXXG domain for
ATP binding, which is present in mERK5a (underlined in Fig.
2A).
Further characterization of the mouse ERK5 protein sequence with
currently available profile data bases resulted in the identification of a proline-rich region and a bipartite nuclear localization signal
(NLS) in the C-terminal domain (Fig. 2B). The proline-rich region and the NLS are located at aa 578-690 and aa 505-539,
respectively. Comparison of the deduced amino acid sequence of mouse
ERK5a with human ERK5 showed 91% homology to human ERK5 (Fig.
2B). Many functional domains important for kinase activity,
including the TEY phosphorylation site, are conserved between human and
mouse ERK5. The major differences between human and mouse ERK5 occur in
a small portion of the N terminus and the proline-rich region in the C terminus.
Presence of Mouse ERK5 Splice Variant Expression in Mouse Embryo
and Adult Mouse Tissues--
To confirm the existence of the three
forms of mouse ERK5 mRNAs, we performed RT-PCR with a different
source of mouse embryo mRNA. Three bands corresponding to PCR
products from the three recombinant cDNAs were detected (Fig.
3A). To determine the presence of three mouse ERK5 mRNAs in adult mouse tissues and examine
tissue-specific expression, RT-PCR was performed for multiple adult
mouse tissues. PCR products encoding the three mouse ERK5 mRNAs
were detected in all mouse adult tissues examined (Fig.
3B).
To confirm the presence of the endogenous protein products
corresponding to the three splice variants, we used a polyclonal antibody against ERK5, which was made using amino acids
EGHGMNPADIESLQREIQMDSPML of the human ERK5 as antigen. This human ERK5
peptide is 100% similar to the corresponding amino acids of the three
mouse isoforms. Preliminary data showed that it recognized mouse and
human ERK5 equally well. Immunoblotting of mouse embryonic proteins
revealed three distinct bands (Fig. 3C) with the molecular
weights corresponding to mERK5a, mERK5b, and mERK5c, respectively.
Relative protein levels of three splice variants are consistent with
their relative mRNA levels, mERK5a > mERK5c > mERK5b.
Kinase Activities and Effects of Different Mouse ERK5 Isoforms on
Transactivation of the Transcription Factor MEF2C--
Because mERK5b
and mERK5c lack the GXGXXG domain required for
ATP binding, it is very likely that mERK5b and mERK5c have no kinase
activity. To further explore the function of mERK5b and mERK5c,
phosphorylation of myelin basic protein (MBP) was evaluated by an
in vitro kinase assay. Immunoprecipitated mERK5a, isolated from the cells coexpressing constitutively active MEK5(D), rapidly phosphorylated MBP (Fig. 4). In contrast,
mERK5b and mERK5c failed to phosphorylate MBP, which is similar to
results using human dominant negative ERK5 (DN-hERK5) (14). To
investigate the possibility that mERK5b and mERK5c act as dominant
negative isoforms, mERK5b or mERK5c were cotransfected with mERK5a and
kinase assays were performed. As shown in the last two lanes
of Fig. 4, mERK5b inhibited mERK5a kinase activity. mERK5c behaved in a
similar manner (data not shown).
Because the transactivation of the transcription factor MEF2C is
stimulated by human ERK5-induced phosphorylation (14), we measured
MEF2C activity as a means to determine the kinase activities of
different ERK5 isoforms. Utilizing fusion proteins containing the
transactivation domain of MEF2C fused to the DNA binding domain of the
yeast transcription factor GAL4, we were able to assess the effects of
the mouse ERK5 isoforms on the activity of transcription factor MEF2C
fusion protein. This was done by measuring the luciferase activity from
CHO-K1 cells cotransfected with a construct containing five copies of
the GAL4-binding site upstream of a luciferase reporter gene. As
expected, MEF2C-dependent reporter gene expression was
enhanced dramatically when mERK5a and MEK5(D) were cotransfected into
CHO-K1 cells (Fig. 5A). mERK5b and mERK5c, similar to DN-hERK5, did not stimulate MEF2C activity (Fig.
5A). The same results were observed after extracellular stimulation with 10% serum (Fig. 5B). To further
demonstrate that mERK5b and mERK5c behave as dominant negative forms,
we coexpressed mERK5b, mERK5c, or DN-hERK5 with mERK5a. Introduction of
mERK5b (Fig. 5C) and mERK5c (Fig. 5D), similar to
DN-hERK5 (Fig. 5E), inhibited mERK5a-induced
MEF2C-dependent reporter gene expression in a
dose-dependent manner. mERK5b and mERK5c also
dose-dependently inhibited MEF2C activation by endogenous
ERK5 after stimulation with 10% serum (Fig. 5F). These
results suggest that mERK5b and mERK5c may function as dominant
negative inhibitors of the ERK5 signaling pathway.
Subcellular Localization of Mouse ERK5 Isoforms--
To determine
the role of the N-terminal region, which is absent in mERK5b and
mERK5c, we examined the subcellular distribution of different ERK5
isoforms by cell fractionation and Western blotting of lysates from
cells expressing epitope-tagged mERK5s. In the unstimulated cells, we
observed that mouse mERK5a and human hERK5 were present in both the
cytoplasm and the nucleus with the majority in the cytoplasm (Fig.
6). However, mERK5b and mERK5c were
exclusively present in the nucleus. There was no detectable mERK5a,
mERK5b, and mERK5c expression in the membrane and cytoskeleton
fractions (data not shown). These results suggest that the N-terminal
domain spanning aa 1-77 is important for cytoplasmic targeting of
mERK5a.
The C-terminal Tail of ERK5 Is Not Required for Catalytic Activity
but Is Essential for Activation of Transcription Factor
MEF2C--
Because the protein sequence analysis of ERK5 uncovered
several interesting domains, we further investigated the roles of these
regions. ERK5 has a unique 400-amino acid long C-terminal tail whose
function is not known. To determine whether the C-terminal domain of
ERK5 plays a role in regulating ERK5 kinase activity, an Xpress-tagged
ERK5 truncated at Gln-406, termed mERK5a(-tail), was generated. When
mERK5a(-tail) and ERK2 amino acid sequences are aligned, the length of
mERK5a(-tail) is comparable to that of ERK2. To test whether
mERK5a(-tail) was catalytically active, an in vitro kinase
assay using MBP as a substrate was performed. Using MEK5(D) to activate
ERK5, mERK5a(-tail) was able to phosphorylate MBP in vitro
similar to mERK5a (Fig. 7A,
upper panel). These results suggest that the kinase activity
of mERK5a(-tail) is comparable to mERK5a.
Another indication of kinase activity is autophosphorylation. The
anti-Xpress antibody recognized both the full-length mERK5A and the
truncated mERK5a(-tail) in cell lysates from transfected CHO-K1 cells,
suggesting that mERK5a(-tail) protein was stably expressed (Fig.
7A, lower panel). Upon activation by coexpression of MEK5(D), mERK5a(-tail) exhibited an electrophoretically shifted band
similar to the full-length mERK5a (Fig. 7A, lower
panel), suggesting that mERK5a(-tail) was phosphorylated by MEK5.
The shifted band of the full-length ERK5 is consistent with the
phosphorylation of ERK5 and is generally thought to correspond to the
activated form (14, 15). Together, these data indicate that the
C-terminal tail is not required for the full kinase activity of ERK5
stimulated with activated MEK5. This result is consistent with the
previous observation that the C-terminal domain was not needed for
kinase activity in vitro (9).
Finally the effects of mERK5a(-tail) on the activity of transcription
factor MEF2C fusion protein were assessed by measuring the luciferase
activity from CHO-K1 cells cotransfected with a construct containing
five copies of the GAL4-binding site upstream of a luciferase reporter
gene. As expected, MEF2C-dependent reporter gene expression
was enhanced dramatically when the full-length mERK5a and MEK5(D) were
cotransfected into CHO-K1 cells (Fig. 7B). However, the
mERK5a(-tail), which can act as an active kinase, did not stimulate
MEF2C activity (Fig. 7B).
The C-terminal Tail Containing the NLS Plays a Role in Nuclear
Translocation of ERK5--
Like other MAPKs, ERK5 is localized in
cytoplasm in the unstimulated state and translocates into the nucleus
upon activation (14). A possible role for the C-terminal domain is to
facilitate the translocation of activated ERK5 to the nucleus, because
this domain is required for the transactivation of the transcription factor MEF2C and it has a possible nuclear localization sequence. To
explore this possibility, immunocytochemistry was performed to test
whether the C-terminal domain influences the nuclear translocation of
mERK5a. In the absence of MEK5(D), cells expressing mERK5a or
mERK5a(-tail) had predominantly cytoplasmic staining (Fig. 8, A and C). In the
presence of MEK5(D), a significant amount of mERK5a localized in the
nucleus (Fig. 8B). In contrast, mERK5a(-tail) was unable to
translocate to the nucleus (Fig. 8D). In CHO cells, recombinant GFP was distributed in both the cytoplasm and the nucleus
(Fig. 8E). However, GFP-tail, a fusion protein containing the GFP and the C-tail of mERK5a (aa 407-806), was exclusively localized in the nucleus (Fig. 8F), suggesting that the
C-tail of mERK5a is able to drive GFP to the nucleus.
Searching the mouse ERK5 sequence against a currently available profile
data base, a putative bipartite nuclear localization signal (NLS) (aa
505-539) was found in the C-tail. To determine whether the putative
NLS is biologically important, GFP-NLS, a fusion protein containing GFP
and the NLS domain of mERK5a (aa 505-539), as well as mERK5a(-NLS), an
NLS-lacking mutant of mERK5a (deleting aa 505-539), were constructed.
As expected, the NLS domain was able to drive GFP to the nucleus (data
not shown). In contrast, mERK5a(-NLS), lacking the NLS domain, was
localized in the cytoplasm under unstimulated conditions (Fig.
8G) but failed to move to the nucleus efficiently upon
activation by MEK5(D) (Fig. 8H), indicating that the NLS
domain is biologically active (Fig. 8, compare B and
H). Taken together, these findings suggest that
translocation into nucleus via the NLS in the C-terminal tail is
essential for biological activity of ERK5 in vivo.
The N-terminal Domain of ERK5 Is Responsible for Its
Oligomerization and Association with MEK5--
It has been reported
that ERK1/2 oligomerizes upon phosphorylation. To determine whether
ERK5 oligomerizes, we coexpressed Xpress-tagged mouse ERK5a (Xp-mERK5a)
and FLAG-tagged mouse ERK5a (FLAG-mERK5a) in CHO-K1 cells and
performed coimmunoprecipitation assays. Immunoprecipitation of
Xp-mERK5a with the Xpress antibody brought down FLAG-mERK5a (Fig.
9A) and vice versa
(data not shown), suggesting that ERK5a forms oligomers in the cells.
Oligomerization of ERK5 was observed in cell lysates from both
activated and control cells, because coimmunoprecipitation was found in
cells with or without expression of MEK5D, different from reported data
for ERK1/2 (20). The observation, that the wild type FLAG-mERK5a and
truncated Xp-mERK5a(-tail) could also be coimmunoprecipitated (Fig.
9B) but tail-GFP could not be coimmunoprecipitated (data not
shown), suggests that the N-terminal domain but not the C-terminal domain of ERK5 is involved in the oligomerization. Furthermore, the
N-terminal truncated isoforms, mERK5b and mERK5c, were able to be
coimmunoprecipitated with wild type mERK5a (Fig. 9B),
suggesting that the region aa 140-406 but not the region aa 1-139 of
the N terminus of ERK5 is important for the oligomerization.
The ability of the N-terminal or C-terminal truncated ERK5 mutants to
bind to MEK5 was also examined by coimmunoprecipitation. The
observation that mERK5a(-tail) and mERK5b but not mERK5c were able to
bind to MEK5 (Fig. 10) indicates that
the region aa 78-139 in the N-terminal domain is important for the
association of ERK5 with MEK5.
In this report we have identified three differentially spliced
mouse ERK5 cDNAs (mERK5a, mERK5b, and mERK5c), which appear to play
unique functional roles in regulating ERK5 and MEF2C activity. mERK5b
and mERK5c function as dominant negative kinases blocking mERK5a
activity and ERK5-mediated MEF2C activation. In addition, we have
investigated the functional roles of several regions of mouse ERK5,
which appear to be important for protein-protein interactions and
intracellular localization. Specifically, we found that the N-terminal
domain aa 1-77 is important for the cytoplasmic targeting; domain aa
78-139 is required for association with the upstream kinase MEK5; and
domain aa 140-406 is necessary for oligomerization. The C-terminal
tail, which contains a putative NLS, was found to be required for
nuclear translocation of mERK5a upon activation (Fig.
11).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
.
-mercaptoethanol, 0.02% bromphenol blue) and subjected to SDS-PAGE, and proteins were then transferred to
nitrocellulose. The membrane was blocked for 1-2 h at room temperature
with a commercial blocking buffer (Life Technologies, Inc.). The blot
was incubated for 1 h at room temperature with the primary
antibody (anti-Xpress antibody from Invitrogen), followed by incubation
for 1 h with secondary antibody (horseradish
peroxidase-conjugated). Immunoreactive bands were visualized by
chemiluminescence (ECL, Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representations of mouse ERK5
splice variants and genomic structure at the splicing junction.
A, schematic diagram of mouse ERK5 cDNAs.
I-1, I-2, and I-3 refer to the
insertions of DNA sequence. The asterisk indicates the stop
codon. B, schematic diagram of partial genomic DNA flanking
the splicing region of mouse ERK5 gene. The splicing donor
gt sites (D1, D2, and D3) and splicing
acceptor ag sites (A1 and A2) are
underlined. Exon and intron regions are represented by
capital and lowercase letters,
respectively.
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Fig. 2.
Amino acid sequence comparison.
A, N-terminal deduced amino acid sequence comparison among
mERK5a, mERK5b, and mERK5c. The asterisk indicates the ATP
binding domain. B, comparison of the amino acid sequences of
human ERK5 and mouse ERK5a. The deduced amino acid sequence of human
ERK5 (hERK5) was aligned with that of mERK5a using the BLAST program.
The proline-rich region and the nuclear localization signal (NLS) are
underlined. The activating phosphorylation motif
(TEY) is enclosed in the box. Identical residues
between the two sequences are indicated by vertical lines.
Gaps, denoted by periods, are introduced into the sequence
to optimize alignment.
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Fig. 3.
Expression of alternatively spliced ERK5
cDNAs in mouse embryos and adult tissues. RT-PCR and
immunoblotting experiments were carried out as described under
"Material and Methods." Arrows indicated bands of three
mERK5 PCR products. A, mRNA expression in mouse embryo.
Mouse embryo mRNA was examined by RT-PCR using primer pairs around
the splicing junction (lane 2) (see "Material and
Methods"). Lane 1 is a negative control. Lanes
3, 4, and 5 represent three mERK5 PCR
products from three mERK5 cDNAs, using the same primer pairs, which
were used as size markers. B, mRNA expression in
multiple adult tissues. RT-PCR for multiple tissue mRNAs were
carried out as described under "Materials and Methods." Lanes
1, 2, and 3 are PCR products from three
mERK5 cDNAs used as size markers. Lanes 5, 7,
9, 11, and 13 are RT-PCR products
performed in the absence of reverse transcriptase, used as a negative
control for contamination. Lanes 4, 6,
8, 10, and 12 are RT-PCR products for
multiple tissues as indicated. C, protein expression
detected by Western blotting. Lane 1 is total mouse embryo
protein; lanes 2, 3, and 4 are three
expressed recombinant mouse ERK5 proteins, which were used as size
markers.
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Fig. 4.
Kinase activity of different mouse ERK5
splice variants. CHO-K1 cells were transfected with the indicated
Xpress-tagged mouse ERK5 constructs and cotransfected with or without
MEK5(D). Growth-arrested cells were harvested and lysed. ERK5 activity
was analyzed using MBP as a substrate in an immune complex kinase assay
as described under "Materials and Methods." The top
panel is a representative autoradiogram showing ERK5 isoform
kinase activity. The expression levels of recombinant mERK5 isoforms
were assessed by Western blot analysis with anti-Xpress antibody
(Xp) (bottom panel).
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Fig. 5.
Activation of MEF2C by different mouse ERK5
splice variants. CHO-K1 cells were cotransfected with the
indicated ERK5 constructs and the reporter plasmid pG5E1bLuc along with
GAL4 fusion expression vectors containing MEF2C in the presence or
absence of MEK5(D). After 24 h, the medium was changed to
serum-free DMEM/F-12 for an additional 24 h. Then cells were
collected for luciferase assay (A, C,
D, E). In the cases of testing the endogenous
ERK5 (B, F), 10% serum was added and the cells
were incubated for an additional 4 h before harvesting.
Transfection efficiency was determined by cotransfection with a green
fluorescence protein (GFP) expression vector (pEGFP-N1, from
CLONTECH). The luciferase activities were
normalized against cells transfected with pG5E1bLuc and GAL4 reporter
plasmid alone, whose value was taken as 1.
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Fig. 6.
Subcellular localizations of different mouse
ERK5 splice variants CHO-K1 cells were transfected with the
indicated Xpress-tagged mouse ERK5 constructs and cellular fractionates
(see "Material and Methods") were subjected to SDS-PAGE followed by
Western blot analysis with anti-Xpress antibody.
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Fig. 7.
The effect of the C-terminal domain on ERK5
activity. A, kinase activity. CHO-K1 cells were
transfected with the indicated Xpress-tagged ERK5 constructs and
cotransfected with or without MEK5(D). Growth-arrested cells were
harvested and lysed. ERK5 activity was analyzed using MBP as a
substrate in an immune complex kinase assay (upper panel).
The expression levels of recombinant mERK5 isoforms were assessed by
Western blot analysis with anti-Xpress antibody (lower
panel). B, activation of MEF2C. CHO-K1 cells were
cotransfected with the indicated ERK5 constructs and the reporter
plasmid pG5E1bLuc along with GAL4 fusion expression vectors containing
MEF2C in the presence or absence of MEK5(D). After 24 h, the
medium was changed to serum-free DMEM/F-12 for an additional 24 h.
Then cells were collected for luciferase assay. Transfection efficiency
was determined by cotransfection with a green fluorescence protein
(GFP) expression vector (pEGFP-N1, from
CLONTECH). The luciferase activities were
normalized against cells transfected with pG5E1bLuc and GAL4 reporter
plasmid alone, whose value was taken as 1.
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Fig. 8.
The effect of the C-terminal domain on
nuclear targeting of ERK5. CHO-K1 cells were cotransfected with
mERK5a (A and B), mERK5a(-tail) (C and
D), or mERK5a(-NLS) (G and H) in the
presence of pCDNA3 vector (A, C, and
G) or MEK5(D) (B, D, and
H). Immunofluorescence staining was performed with the
anti-Xpress antibody as a first antibody and a fluorescein-conjugated
anti-mouse IgG as a secondary antibody. CHO-K1 cells were transfected
with pEGFP-C3 purchased from CLONTECH
(E) or GFP-tail containing the C-terminal tail of mERK5a
fused to the C terminus of EGFP (F). The images were
analyzed and captured with an Olympus Fluoview confocal
microscope.
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Fig. 9.
The effects of N-terminal regions of mouse
ERK5 on the oligomerization of ERK5. CHO-K1 cells were transfected
with the indicated Xpress-tagged mERK5a (A), mERK5b
(B), mERK5c (B), or mERK5a(-tail) (B)
together with FLAG-tagged mERK5a in the presence or absence of MEK5(D).
Growth-arrested cells were harvested and lysed. Xpress-tagged mouse
ERK5 proteins were immunoprecipitated and analyzed by SDS-PAGE.
Immunoblotting with either anti-Xpress or anti-FLAG antibody was
performed to demonstrate the occurrence of coimmunoprecipitation.
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Fig. 10.
The effects of N-terminal regions of mouse
ERK5 on the association with MEK5. CHO-K1 cells were cotransfected
with the indicated Xpress-tagged mouse ERK5 constructs together with or
without HA-tagged MEK5(D). Growth-arrested cells were harvested and
lysed. Xpress-tagged mouse ERK5 proteins were immunoprecipitated and
analyzed by SDS-PAGE. Immunoblotting with anti-HA antibody was
performed to demonstrate the occurrence of coimmunoprecipitation
between ERK5 and MEK5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Schematic diagram showing the organization
of functional domains.
We identified three mouse ERK5 cDNAs by homology analysis, mERK5a (a mouse homologue of human ERK5), and two truncated mouse ERK5 isoforms termed mERK5b and mERK5c. Analysis of mouse genomic DNA sequences adjacent to the splicing junctions suggests that the three mERK5 cDNAs are generated by alternative splicing using different splicing donors and acceptors from a single gene encoding mouse ERK5. Several different ERK5 transcripts have been shown in human tissue (8), but these were generated by alternative splicing occurring at the 5'-noncoding region. The sequence of mERK5a is the same as that of the mouse ERK5 reported previously by Kamakura et al. (21) except for six scattered amino acid mismatches within the entire open reading frame. Possible explanations are a sequencing error or a DNA polymorphism causing the sequence variation.
The most abundant cDNA was mERK5a, which shares 91% identity with human ERK5. Compared with mERK5a, mERK5b and mERK5c lacked 69 and 139 amino acids at their N terminus, respectively. It appears likely that mERK5b and mERK5c perform functions different from mERK5a, because these proteins are unable to bind ATP and therefore are not active kinases. Our observations, that mERK5b and mERK5c lack kinase activity, inhibit mERK5a kinase activity, and inhibit mERK5a-mediated MEF2C transactivation, suggest that mERK5b and mERK5c may act as endogenous dominant negative kinases if mERK5b and mERK5c are expressed endogenously to a significant extent under some conditions. Immunoblotting results indicate that mERK5a is expressed to a greater extent than mERK5c and much greater than mERK5b. It is possible that the proteins encoded by mERK5b and mERK5c mRNAs could be expressed to different extents under some conditions or selectively expressed in some cell types. It is also possible that the mRNAs of mERK5b and mERK5c are not efficiently translated to the protein products in vivo as predicted, because the ribosome my initiate and terminate the translation early by the stop codon in the first insert. Thus, the expression of alternatively spliced mERK5b and mERK5c mRNAs may provide a mechanism to regulate ERK5 protein expression by preventing the translation of ERK5 in some cells at specific developmental stages or pathological conditions. Future studies are necessary to clarify the biological significance of mERK5b and mERK5c mRNAs in the regulation of mERK5a function.
Several other kinases exhibit independent expression of noncatalytic domains resulting from alternative splicing, which function as endogenous dominant negative inhibitors. For example, the primary transcript of a calmodulin-dependent protein kinase is alternatively spliced to generate mRNAs encoding either the full-length kinase or the calmodulin binding domain alone (22). Focal adhesion-associated protein tyrosine kinase (FAK) has an independent, C-terminal, noncatalytic domain (FRNK, FAK-related nonkinase) (23). Finally, the C terminus of the smooth muscle myosin light chain kinase is also expressed as an independent protein, telokin (24). Thus, the truncated, catalytically inactive forms of the mouse ERK5, mERK5b, and mERK5c may function as endogenous dominant negative inhibitors if they are expressed endogenously to a significant extent under some conditions.
Searching the mouse ERK5 sequence against a currently available profile data base to identify known functional regions, a proline-rich region and a NLS were found in the C-terminal domain. Proline-rich regions exist widely in both prokaryotes and eukaryotes. Studies have shown that proline-rich regions may act as Src-homology 3-binding motifs. These regions can directly interact with other proteins containing Src-homology 3 domains to regulate cellular localization and/or modulate enzymatic properties (1, 2). A proline-rich sequence unique to MEK1 and MEK2 is required for Raf binding and MEK function (25). The function of proline-rich regions remains largely unclear. Interestingly, a significant difference between mouse ERK5a and human ERK5 sequences is present in the proline-rich region. Thus, the cloned mouse ERK5a, which differs significantly only in the proline-rich region compared with the human ERK5, may be a useful gene to determine the function of proline-rich regions. Differences between human and mouse with regard to intracellular signal transduction by highly related proteins have been reported (26, 27). For example, the BAS-like Fas-associated phosphatase-1 interacts with the human Fas receptor, but not with the mouse Fas receptor. The C terminus of the Fas receptor, which is required for this interaction, is not conserved between mouse and human. Elucidation of the role of the mouse ERK5 proline-rich region may also reveal differences in signaling mechanisms between human and mouse.
The putative bipartite NLS in ERK5 located in the C-terminal tail is very likely to be important for nuclear translocation of ERK5. A bipartite NLS was described initially in the nucleosome assembly factor nucleoplasmin, which consists of two basic amino acids, a spacer region of any 10-12 amino acids, and a basic cluster in which at least three out of the next five amino acids must be basic (28). Although the bipartite motif is a considerably more reliable indicator of nuclear localization, because less than 5% of non-nuclear proteins have a sequence that fits this motif (29), it is important to demonstrate that the putative NLS is necessary for nuclear targeting of the parent protein and sufficient to direct a non-nuclear protein to the nucleus. Our observations, that the NLS of mERK5 is required for the nuclear targeting of mERK5 upon activation and that this NLS itself is sufficient to drive GFP to the nucleus, indicate that the mERK5 NLS is biologically functional.
The mechanisms for the nuclear translocation and cytoplasmic anchoring of ERK1/2 must be different from ERK5. It has been suggested that ERK1/2 could cross the nuclear envelope by passive diffusion if it does not have either a NLS or a nuclear export signal. The cytoplasm retention of ERK1/2 in unstimulated cells likely involves specific association with MEK1/2, and nuclear translocation of ERK1/2 upon stimulation is accompanied by dissociation from MEK1/2 (30). A nuclear export signal in the N terminus of MEK1/2 has also been identified (31). These data indicate that MEK1/2 is a cytoplasmic anchoring protein for ERK1/2. The molecular size of ERK5 is beyond the limit for passive diffusion through the nuclear envelope pore. The nuclear targeting of ERK5 seems to be mediated by the NLS. The observation that mERK5b or mERK5c was present in the nuclear portion suggest that the N-terminal domain aa 1-77 is responsible for the cytoplasmic localization. The role for the N-terminal domain aa 1-77 in cytoplasmic localization of mERK5 is not clear. It is possible that the N-terminal domain aa 1-77 associates with other cytoplasmic components other than MEK5.
ERK5, unlike ERK1/2, exists as oligomer in unstimulated cells. ERK1/2 oligomerizes in a phosphorylation-dependent manner (32). In addition, ERK1/2 dimers are composed of either two phosphorylated molecules or one phosphorylated and one unphosphorylated molecule. In contrast, ERK5 expressed from transfected plasmids was able to form oligomers in both stimulated and unstimulated conditions, suggesting that the oligomerization of ERK5 is not dependent on its phosphorylation status. We found that the region aa 140-406 in ERK5 is important for oligomerization, homologous to the dimer interface of ERK1/2, which is localized to amino acids 170-359 (32).
Among the MAPKs, ERK1/2, ERK5, and ERK7 share the same signature TEY activation motif. However, these three MAPKs are completely different in terms of their structure, activation, and mechanisms of regulation, mediated in part by the fact that ERK5 and ERK7 have unique C-terminal tails compared with ERK1/2. Activation by TEY phosphorylation of ERK2 leads to nuclear import even though ERK1/2 does not have any classic nuclear localization signal. It has been suggested that interaction of ERK1/2 with upstream kinases may control nucleocytoplasmic transport (30). ERK7 is a constitutively activated and permanently nuclear-localized enzyme. Its activation and kinase activity seem to be dependent on nuclear targeting, which requires the C-terminal tail (11). In the case of ERK5, nuclear translocation occurs upon activation. However, its activation and kinetic activity are not dependent on its C-terminal tail.
In summary, we have identified three differentially spliced mouse ERK5
cDNAs whose unique structures result in different roles in the
regulation of ERK5 and MEF2C activity. Using these mouse ERK5 splicing
variants and other constructed mutants, we have also located regions of
ERK5 that are responsible for cytoplasmic targeting, nuclear
translocation, oligomerization, and MEK5 binding. Further studies are
required to characterize in detail the precise role of these three
isoforms in the specific function of ERK5.
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ACKNOWLEDGEMENT |
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We thank James Surapisitchat for critical reading and corrections.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL49192 and HL18645 (to B. C. B.).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.
§ Both authors contributed equally to this work.
¶ Recipient of National Institutes of Health Cardiovascular Training Grant T32HL07828.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF126159 (mERK5a), AF126160 (mERK5b), and AF126161 (mERK5c).
To whom correspondence should be addressed: Center for
Cardiovascular Research, Box 679, 601 Elmwood Ave., University of
Rochester School of Medicine and Dentistry, Rochester, NY 14642. Tel.:
716-273-1946; Fax: 716-273-1497; E-mail:
bradford_berk@urmc.rochester.edu.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M009286200
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPK kinase kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; MEK, MAPK/ERK kinase; NLS, nuclear localization signal; aa, amino acid(s); JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; BMK1, Big MAP kinase 1 (also known as ERK5); EGF, epidermal growth factor; EST, expressed sequence tag; PCR, polymerase chain reaction; RT, reverse transcription; GFP, green fluorescence protein; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagles' medium; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); MBP, myelin basic protein; DN, dominant negative; FAK, focal adhesion-associated protein tyrosine kinase.
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