Oncogene (1997) 15, 677 ± 683
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
The protooncogene product, PEBP2b/CBFb, is mainly located in the
cytoplasm and has an anity with cytoskeletal structures
Yuta Tanaka1,2, Toshio Watanabe1, Natsuko Chiba1, Masaru Niki1, Yasuyuki Kuroiwa3,
Tetsuro Nishihira2, Susumu Satomi2, Yoshiaki Ito4 and Masanobu Satake1
1
Department of Molecular Immunology, Institute of Development, Aging and Cancer; 2The Second Department of Surgery, School
of Medicine, Tohoku University, Seiryo-machi, Aoba-ku, Sendai 980 ± 77; 3Pharmaceutical Research Laboratory, Hitachi Chemical
Co., Ltd., Higashi-cho, Hitachi 317; 4Department of Viral Oncology, Institute for Virus Research, Kyoto University, Shogoin,
Sakyo-ku, Kyoto 606 ± 01, Japan
The Pebpb2/Cbfb gene encodes the non-DNA binding b
subunit of the heterodimeric transcription factor,
PEBP2/CBF, and has been implicated in a subtype of
human acute myeloid leukemia, as well as being
indispensable for the development of de®nitive hematopoiesis in the murine fetal liver. By examining a
subcellular localization of the PEBP2b/CBFb protein
in tissue culture cells, we could reveal an additional
aspect of the protein other than to be a subunit of a
transcription factor. Immunoblot and immunocytochemical staining showed that PEBP2b/CBFb was mostly
present in the cytoplasm. This PEBP2b/CBFb was free
from its DNA-binding partner, the a subunit of PEBP2/
CBF, as judged by the electrophoretic mobility shift
assays. Furthermore, a signi®cant amount of PEBP2b/
CBFb was retained in the cytoskeleton preparation after
detergent extraction of the cells and was found by double
immuno¯uorescence to colocalize with the F-actin on
stress ®bers and the vinculin in membrane processes.
Thus, the present study extends PEBP2b/CBFb to be a
cytoskeleton-anitive as well as nuclear protein. The
implications of these results are discussed.
Keywords: PEBP2b/CBFb; proto-oncogene; acute
myeloid leukemia; transcription factor; cytoskeleton;
immunocytochemistry
Introduction
The transcription factor PEBP2/CBF was originally
identi®ed as an element that bound to the enhancers of
murine polyomavirus and leukemia virus, and was
subsequently shown to be a heterodimer composed of a
and b subunits. (Kamachi et al., 1990; Ogawa et al.,
1993a,b; Wang and Speck, 1992; Wang et al., 1993).
The Runt domain, which is conserved among the a
subunit-encoding genes, is responsible for DNA
binding activity and heterodimer formation with the
b polypeptide (Bae et al., 1994; Kagoshima et al., 1993;
Meyers et al., 1993; Ogawa et al., 1993b). On the other
hand, the b subunit dimerizes with the a subunit to
enhance its DNA binding anity, but does not show
any intrinsic DNA-binding activity by itself (Ogawa et
al., 1993a; Wang et al., 1993). Of pathological
importance is the fact that both the a subunitencoding Aml1/Pebpa2b/Cbfa2 and the b subunitCorrespondence: M Satake
Received 18 December 1996; revised 25 April 1997; accepted 25 April
1997
encoding Pebpb2/Cbfb are protooncogenes involved in
human acute myeloid and/or lymphoblastic leukemia
(Erickson et al., 1992; Golub et al., 1995; Liu et al.,
1993; Mitani et al., 1994; Miyoshi et al., 1991;
Nucifora et al., 1993; Nucifora and Rowley, 1995;
Romana et al., 1995; Shurtle€ et al., 1995). The genes
are located at the breakpoints in several types of
chromosomal translocations and/or inversion associated with these leukemia. The notion that the a and
b subunits function together in vivo as a complex
termed PEBP2/CBF has been demonstrated in a
remarkable way by recent gene targeting studies.
Targeting of Pebpb2/Cbfb showed that this gene is
essential for the development of de®nitive hematopoiesis in the murine fetal liver (Sasaki et al., 1996; Wang
et al., 1996b; Niki et al., 1997). The Pebpb2/Cbfb (7/
7) embryos did not develop the multipotent hematopoietic progenitors in the liver and died on embryonic
day 12.5 probably as a result of hemorrhaging into the
central nervous system. An almost identical phenotype
was observed after the targeting of Aml1/Pebpa2b/
Cbfa2 (Okuda et al., 1996; Wang et al., 1996a).
A prerequisite for PEBP2/CBF to function as a
transcription factor is its location in the nucleus. As far
as the expression and localization of the PEBP2b/
CBFb polypeptide are concerned, there are, however,
several unexpected observations from the fact that
PEBP2b/CBFb is a subunit of a transcription factor.
The ®rst of these was our report dealing with the
subcellular localization of the a and b proteins as
examined by immunochemical staining of c-DNA
transfected NIH3T3 cells (Lu et al., 1995). In such
cells, the a protein alone was found to be localized in
the nucleus, whereas the b protein alone was localized
in the cytoplasm. Only when the b was coexpressed
with a truncated form of the a protein, did the b
subunit become detectable in the nucleus. Secondly, we
recently observed by immunohistochemistry that the
PEBP2b/CBFb was expressed in a di€erentiation
dependent manner in the skeletal myogenic cells of
murine embryos, and that the protein mainly
accumulated in the cytoplasm (Chiba et al., 1997).
Only during a limited stage of di€erentiation, namely
in the multinucleated myotubes, was nuclear staining
observed for PEBP2b/CBFb. These two observations,
therefore, indicate that the localization of PEBP2b/
CBFb can be just as well cytoplasmic as nuclear.
One of the interesting aspects of the cytoplasmic
PEBP2b/CBFb is our immunohistochemical observation that the protein was colocalized with the a-actinin
on the Z-lines or their vicinity in the established
skeletal muscle ®bers (Chiba et al., 1997). This
observation suggests an anity of PEBP2b/CBFb with
cytoskeletal structures. In the present study, we have
examined such a possibility using tissue culture cells.
We demonstrate that the cytoplasmic PEBP2b/CBFb is
in a form that is free from the a subunit, and that a
part of this form is retained in the cytoskeleton
preparation after detergent extraction of the cells and
colocalizes with stress ®bers.
βcDNA:
CSK
678
SOL
Affinity of PEBP2b/CBFb with the cytoskeleton
Y Tanaka et al
–
+
–
+
2
3
4
5
kDa
175 —
83 —
62 —
47.5 —
Results
Colocalization of PEBP2b/CBFb with stress ®bers on
the cytoskeleton preparation
A monolayer culture of REF52 cells was treated
gently with 0.1% Nonidet P-40 (NP-40) in a
microtubule stabilizing bu€er and fractionated into
soluble and cytoskeleton preparations. The amounts
of proteins from each fraction which corresponded to
the equivalent cell number were separated by SDSpolyacrylamide gel electrophoresis (PAGE) and
blotted onto a membrane. The ®lter was probed with
anti-b peptide antibody (Figure 1) whose preparation
and speci®city were described previously (Chiba et al.,
1997). As seen in lane 4, a signi®cant proportion of
the 22 kilodaltons PEBP2b/CBFb protein which were
expressed endogenously in the cells was found to be
retained in the cytoskeleton fraction. On the other
hand, only a relatively small amount of the protein
was released into the soluble fraction (lane 2).
Retention of PEBP2b/CBFb in the cytoskeleton
preparation was also observed for the protein
expressed exogenously from the transfected c-DNA
(compare lanes 3 and 5). Thus, the immunoblot data
indicate that a considerable proportion of PEBP2b/
CBFb, irrespective of whether it was expressed
endogenously or from the transfected c-DNA, is
retained on cytoskeletal structures. The extraction
procedure used above was con®rmed to be adequate,
since most of the lactate dehydrogenase molecules was
recovered in the soluble fraction, whereas most of the
actin molecules was retained in the cytoskeleton
fraction as revealed by an immunoblot analysis using
respective antibodies (data not shown).
We next examined the morphological distribution of
PEBP2b/CBFb on the cytoskeleton preparation by
immunocytochemistry. REF52 cells transfected by the
Pebpb2/Cbfb cDNA were extracted with detergent as
described above, ®xed and processed for double
immuno¯uorescent staining (Figure 2). PEBP2b/CBFb
was detected by the anti-peptide antibody followed by
the ¯uorescein-isothiocyanate (FITC) conjugated secondary antibody (Figure 2a), whereas F-actin was
detected by rhodamine-isothiocyanate (RITC) conjugated phalloidin (Figure 2b). The samples were viewed
through a confocal laser scanning microscope. Figure
2c is a superimposed image where a yellow color
signi®es colocalization of ¯uorescence from FITC and
RITC. PEBP2b/CBFb was retained in the cytoskeleton
preparation and showed some degree of colocalization
with F-actin containing stress ®bers (examples are
indicated by arrowheads). In the above experiments,
speci®city of the PEBP2b/CBFb staining was con®rmed by preabsorption of the antibody with the
peptide (data not shown).
32.5 —
25 —
16.5 —
6.5 —
1
Figure 1 Immunoblot detection of PEBP2b/CBFb in the
cytoskeleton fraction of REF52 cells. A plasmid expressing the
b2 isoform of PEBP2b/CBFb was transfected (lanes 3 and 5) or
not (lanes 2 and 4) into REF52 cells. The cells were treated by
the detergent and the soluble (lanes 2 and 3) and cytoskeletal
fractions (lanes 4 and 5) were prepared. Twenty mg of the
protein from each fraction was subjected to SDS-PAGE and the
blotted ®lter was incubated with the anti-b peptide antibody and
processed for immunodetection. The bands indicated by an
arrowhead represent the PEBP2b/CBFb protein. The other
bands are considered to be due to non-speci®c reaction, judged
by the preabsorption experiment of the antibody with the
peptide. In lane 1, the total cell extract (10 mg) from mouse C2
myoblasts was run for comparison. This cell line expresses a
relatively larger amount of PEBP2b/CBFb, compared to REF52
and NIH3T3 cells. Molecular weight markers are shown as
kilodaltons
Stress ®bers that run in parallel with the longuitudinal axis of the cell, on reaching the cell periphery,
lodge into the cell membrane via molecules including
the vinculin (Burridge et al., 1988; Geiger, 1979, 1989).
Double labelling was performed as above except that
the anti-vinculin antibody was used instead of
phalloidin. Figure 2d shows staining for PEBP2b/
CBFb, whereas the staining in Figure 2e represents
vinculin. As seen in the superimposed image in Figure
2f, PEBP2b/CBFb colocalized in some degree with the
vinculin in membrane processes.
To exclude a possibility of an artifact caused by a
detergent extraction procedure, the c-DNA transfected
REF52 cells were ®xed without detergent extraction
and processed for double immuno¯uorescence as
above. PEBP2b/CBFb again displayed colocalization
with the F-actin on stress ®bers or with the vinculin in
membrane processes (data not shown).
Cytoplasmic PEBP2b/CBFb as a free form, away from
the a subunit
The PEBP2/CBF transcription factor is a heterodimer
of the DNA-binding a protein and the non-DNA
binding b protein (Kamachi et al., 1990). It is
reasonable to speculate that the b protein which was
Affinity of PEBP2b/CBFb with the cytoskeleton
Y Tanaka et al
Figure 2 Double immuno¯uorescent staining of PEBP2b/CBFb
and F-actin or vinculin in the cytoskeletal preparation of
REF52 cells. A plasmid expressing the b2 isoform of PEBP2b/
CBFb was transfected into REF52 cells. The cytoskeleton
prepared by detergent extraction of the cells was ®xed and
double labeled for PEBP2b/CBFb and F-actin or vinculin.
PEBP2b/CBFb was detected by the anti-b peptide antibody
followed by the FITC-conjugated secondary antibody (a and d),
whereas F-actin (b) or vinculin (e) were detected by RITCconjugated phalloidin or the RITC-conjugated secondary
antibody, respectively. (c and f) are superimposed images of
¯uorescence from FITC and RITC
retained on the cytoskeleton preparation and colocalized with stress ®bers exists in a free form, away from
the a protein. This possibility was examined by using
the electrophoretic mobility shift assay (EMSA, Figure
3). NIH3T3 cells were used in this assay, since PEBP2/
CBF DNA binding activity has been characterized well
in this cell line (Kamachi et al., 1990).
NIH3T3 cells were transfected by plasmids
expressing individually the Runt domain of AML1/
PEBP2aB/CBFA2, b1, b2 or b3 isoforms of PEBP2b/
CBFb. The three isoforms of PEBP2b/CBFb are
generated by an alternatively splicing mechanism of
the transcripts and di€er in their carboxy-terminal
amino acid sequences (Ogawa et al., 1993a; Wang et
al., 1993). Whole-cell extracts were prepared and
subjected to EMSA, using the PEBP2/CBF binding
sequence as a probe (Figure 3a). The cell extract
receiving the backbone-vector yielded a band of
endogenous PEBP2/CBF DNA binding activity
(lanes 1 and 2). This band represents the ab
heterodimer. The extracts of the cells transfected by
b1, b2 or b3 gave bands with the same mobility as
endogenous PEBP2/CBF (lanes 5 through 10),
indicating that overexpression of b proteins does
not have any apparent e€ect on PEBP2/CBF site
DNA binding activity. On the other hand, the extract
of the cells transfected by the Runt domain yielded
three distinct, new bands each of which corresponded
to Runt alone, a Runt/b1 heterodimer and a Runt/b2
heterodimer, respectively (lanes 3, 4 and 11, see
below for the identi®cation of the new protein/DNA
complexes). The occurrence of the bands, Runt/b1
and Runt/b2, is compatible with the interpretation
that at least some of the endogenous b protein exists
in a free form, away from the a protein, and that
this free fraction is recruited by the exogenously
expressed Runt domain to generate a Runt/b
heterodimer. Presence of a faint band corresponding
to PEBP2/CBF in the Runt-transfected cells indicates
that endogenous PEBP2/CBF remained intact under
these conditions. The sequence speci®c DNA binding
of all the detected protein/DNA complexes was
con®rmed by a competition assay (Figure 3b). The
inclusion of an excessive amount of non-labeled wild
type oligonucleotide abolished the mobility shift of
the DNA bands, whereas mutated oligonucleotide
had no e€ect.
To make sure that the new bands corresponded to
Runt, Runt/b1 and Runt/b2 complexes, we mixed
extracts in vitro and then subjected the mixtures to
EMSA (Figure 3c). The mixing of Runt- and vector
alone-transfected cell extracts did not cause any
essential change in the protein/DNA complexes (lane
2). On the other hand, when the extracts of Runt- and
the extracts of b1- or b2-transfected cells were mixed,
the DNA binding activities corresponding to putative
Runt/b1 or Runt/b2 complexes were greatly enhanced
(lanes 3 and 4). At the same time the band
corresponding to the putative Runt domain-DNA
complex disappeared. The result indicates that the
exogenously expressed b1 or b2 proteins associate with
the Runt domain to yield signi®cantly increased
amounts of Runt/b1 or Runt/b2 heterodimers. A
slight di€erence in the mobilities of the Runt/b1 and
Runt/b2 complexes in the EMSA is in agreement with
the di€erence between the calculated molecular masses
of the b1 and b2 proteins (Ogawa et al., 1993a). The
mixing of the Runt domain extracts and b3 extracts did
not cause any appreciable change (lane 5), in
accordance with our previous report (Ogawa et al.,
1993a).
Finally, we quantitated the relative amounts of the
endogenous PEBP2b/CBFb protein present in the
cytoplasm or the nucleus of NIH3T3 cells by cell
fractionation and immunoblot analysis. The cells
were treated by 1% NP-40 and fractionated by
centrifugation into cytoplasmic and nuclear fractions.
The proteins from each fraction which corresponded
to the same number of cells were separated by gel
electrophoresis and the blotted ®lter was probed with
the anti-b peptide antibody (Figure 4). As seen in
lanes 2 and 3, most of the PEBP2b/CBFb protein
was recovered in the cytoplasmic fraction. When
NIH3T3 cells were immunocytochemically stained,
most of the staining was detected in the cytoplasm
as expected (data not shown). The data of
immunoblot and immunocytochemistry thus demon-
679
Affinity of PEBP2b/CBFb with the cytoskeleton
Y Tanaka et al
680
a
vector
β1
Runt
β2
β3
Runt
PEBP2 —
Runt/β1 —
Runt/β2 —
Runt —
1
2
3
4
5
b
6
7
8
9
10
11
c
+β3
+β2
+β1
+vector
–
Runt
+mutant
+wild
–
Runt
+mutant
+wild
–
vector
— PEBP2
— Runt/β1
— Runt/β2
— Runt
1
2
3
4
5
6
1
2
3
4
5
Figure 3 PEBP2/CBF site DNA binding activities in plasmids-transfected NIH3T3 cells detected by EMSA. NIH3T3 cells were
either transfected with a plasmid expressing the Runt domain of AML1/PEBP2aB/CBFA2, or with plasmids that individually
expressed the b1, b2 or b3 isoforms of PEBP2b/CBFb, as indicated. The cells were also transfected with a backbone plasmid
harboring no cDNA sequence (in lanes indicated as a vector). Whole-cell extracts were prepared and subjected to EMSA. The
amount of extracts corresponded to 2 mg protein in the lanes having odd numbers in (a) and lanes 4 through to 6 in (b). In the even
numbered lanes of (a) and in the lanes 1 through to 3 of (b), 4 mg of protein was used. Lane 11 in (a) is a lightly exposed image of
lane 4. In (b), a 100-fold molar excess of unlabeled wild type PEBP2/CBF site sequence (lanes 2 and 5) or mutated sequence (lanes 3
and 6) was included. In (c) 2 mg protein from the Runt domain-transfected cells was preincubated for 60 min on ice with 2 mg
protein from either the vector alone, b1, b2 or b3 transfected cells and the mixture was then subjected to EMSA
strate that the PEBP2b/CBFb protein expressed
endogenously in NIH3T3 cells was mainly located
in the cytoplasm.
We must note that a PEBP2/CBF DNA binding
activity was detected in non-transfected NIH3T3 cells
(see lane 2 in Figure 3a). Conceivably, an amount of b
protein below a detectable level by immunological
procedures could be associated with the a protein and
be present as a heterodimer in the nucleus of NIH3T3
cells.
Discussion
PEBP2b/CBFb and cytoskeleton
In the present study we have shown that the
endogenous PEBP2b/CBFb in the cell is mainly
present in the cytoplasm. This cytoplasmic PEBP2b/
CBFb is most likely to be in a free form, separate from
its partner, the a subunit, as judged by EMSA. Instead
of interacting with the a protein, the cytoplasmic
Affinity of PEBP2b/CBFb with the cytoskeleton
Y Tanaka et al
kDa
C
N
2
3
175 —
83 —
62 —
147.5 —
32.5 —
25 —
16.5 —
6.5 —
1
Figure 4 Immunoblot detection of PEBP2b/CBFb expressed
endogenously in NIH3T3 cells. The cells were fractionated into
the cytoplasmic (lane 2) and nuclear fractions (lane 3). Fifty ml of
each fraction which contained 50 mg protein in lane 2 and 3.5 mg
protein in lane 3 were separated by SDS ± PAGE and the blotted
®lter was probed with the anti-b peptide antibody. In lane 1, the
total cell extract (10 mg) from C2 myoblasts was run for
comparison. Molecular weight markers are shown as kilodaltons
PEBP2b/CBFb was found to be retained in the
cytoskeleton preparation and colocalize with the Factin on stress ®bers and the vinculin in membrane
processes. The anity between PEBP2b/CBFb and the
cytoskeleton appears relatively weak, however, since
only a part of the PEBP2b/CBFb present in the cell
colocalized with stress ®bers. Most of the b protein
appeared as di€use staining in the cytoplasm. In
addition, when the two c-DNAs were coexpressed,
relocalization of the b protein to the nucleus in the
presence of a truncated form of the a protein did not
apparently a€ect the morphology of the cell or the
stress ®bers (data not shown). Therefore, it seems
unlikely that PEBP2b/CBFb is an authentic component
that constitutes by itself part of the stress ®bers. Such a
relatively weak anity may be related to the fact that a
modest level of homology exists between PEBP2b/
CBFb and cytoskeletal proteins such as troponin T and
non-muscle myosin heavy chain (data not shown).
Implication of the cytoskeleton-anitive PEBP2b/CBFb
What kind of a role can be inferred for the
cytoskeleton-anitive PEBP2b/CBFb? The NFkB/IkB
complex presents a somewhat analogous precedent that
also involves translocation from the cytoskeleton to the
nucleus. Under unstimulated conditions NFkB is
retained in the cytoplasm as a complex with the
inhibitor protein, IkB, which has an ankyryn repeat
(Baeuerle and Baltimore, 1988). Phosphorylation,
ubiquitination and degradation of IkB results in
NFkB release from this complex and translocation of
NFkB into the nucleus (Palombella et al., 1994).
Another case is the glucocorticoid receptor which
forms a complex with hsp70 and hsp90 and is located
in the cytoplasm (see a review by Pratt, 1993). The
binding of the ligand to the receptor triggers the
dissociation of the complex and releases the receptor
from the hsp proteins, thus driving the receptor into
the nucleus. In a fashion analogous to IkB or hsp
proteins, the cytoskeleton in the cytoplasm may serve
to anchor PEBP2b/CBFb. Thus, the PEBP2b/CBFb
located on the cytoskeleton may act as a reservoir of
free b before transportation into the nucleus as part of
an ab heterodimer.
On the other hand, the possibility cannot be
excluded that PEBP2b/CBFb plays an yet unidentified
role in the structure/function of cytoskeleton. A
protein that shows the characteristics of both a
cytoskeletal and transcriptional protein is b-catenin.
On the one hand, b-catenin is a component of the
adherens junction and links the cadherin membrane
protein to the cytoskeleton (Aberle et al., 1994;
HuÈlsken et al., 1994; McCrea et al., 1991; Nagafuchi
et al., 1994). On the other hand, in the Wnt-signaling
pathway, formation of a complex of cytoplasmic bcatenin with the lymphoid enhancer-binding factor 1
(LEF-1) results in the translocation of b-catenin into
the nucleus where it alters the DNA bending activity of
LEF-1 (Behrens et al., 1996; Huber et al., 1996).
In inv 16(p13q22) which is involved in a subtype of
human acute myeloid leukemia, the chimeric gene is
generated between the Pebpb2/Cbfb and smooth muscle
myosin heavy chain (SMMHC) genes (Liu et al., 1993). It
should be pointed out that the SMMHC portion in the
chimeric product represents the rod domain of the
myosin heavy chain molecule and that the chimeric
protein, when expressed alone in the c-DNA transfected
NIH3T3 cells, is located in the cytoplasm, as shown in
our previous study (Lu et al., 1995). In the case of chronic
myeloid leukemia, BCR/ABL protein is associated with
actin-micro®laments and is responsible for the enhanced
phosphorylation of focal adhesion proteins (Salgia et al.,
1995). An extension of our present study on the
cytoskeleton-anitive PEBP2b/CBFb to that on the
PEBP2b/CBFb-SMMHC chimeric protein should increase our understanding of the molecular mechanisms of
leukemogenesis caused by inv 16 (p13q22).
Materials and methods
Cell culture and c-DNA transfection
NIH3T3 and REF52 cells were maintained in Dulbecco
modi®ed Eagle medium supplemented with 10% (vol/vol)
calf serum (NIH3T3) or fetal bovine serum (REF52). DNA
transfection was performed by a calcium phosphate coprecipitation method, as modi®ed by Chen and Okayama
(1987). Four or twenty mg DNA was used for cells grown
in 3- or 10 cm-diameter dishes. The expression plasmids
were pEF-b1, pEF-b2, pEF-b3 and pCX2neoBS, all of
which were previously described (Lu et al., 1995; Sakakura
et al., 1994). The former three plasmids express b1, b2 or
b3 isoform of PEBP2b/CBFb, respectively, while
pCX2neoBS harbors the Runt domain of AML1/
PEBP2aB/CBFA2 protein. Fifteen hours after seeding the
cells were transfected with the c-DNA, incubated for a
further 26 h and then processed for immunochemical
analysis or EMSA.
The antibody
The anti-PEBP2b/CBFb peptide antibody was raised in
hens as described previously (Chiba et al., 1997). This
antibody was puri®ed for immunocytochemical staining by
681
Affinity of PEBP2b/CBFb with the cytoskeleton
Y Tanaka et al
682
anity chromatography on a column in which the peptide
was linked to cyanogen bromide-activated Sepharose 4B
(Pharmacia). In a preabsorption experiment, 100 ml of 100fold-diluted, anity puri®ed anti-peptide antibody was
incubated with 20 mg of the peptide on ice for 60 min prior
to the use in immunodetection.
Preparation of the cytoplasmic and nuclear fractions
NIH3T3 cells grown in 10 cm-diameter dishes at 16106
cells were rinsed twice in phosphate bu€ered saline (PBS)
and then scraped into 500 ml of a TNE bu€er containing
1% (vol/vol) NP-40. TNE was 10 m M Tris-HCl, pH 7.5,
150 mM NaCl, 1 mM EDTA and 0.01 mM phenylmethylsulfonyl ¯uoride (PMSF). After centrifugation at
3000 r.p.m. for 5 min at 48C, the supernatant was taken
to be the cytoplasmic fraction. The pellet was dissolved by
rotating for 60 min at 48C in 500 ml of a RIPA bu€er
(10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%
[vol/vol] NP-40, 0.1% [wt/vol] sodium deoxycholate, 0.1%
[wt/vol] SDS, 10 mg/ml of aprotinin, 2.5 mM dithiothreitol
and 1 mM PMSF). After sonication and centrifugation at
15000 r.p.m. for 10 min at 48C, the supernatant was taken
to be the nuclear fraction. The protein from each fraction
was subjected to an immunoblot analysis.
The primary antibody used was 1000-fold-diluted anti-b
peptide chick IgY solution (11.3 mg/ml).
Immunocytochemistry
REF52 cells were seeded at 16105 cells per 3 cm-diameter
dish, into which glass coverslips had already been placed.
The cytoskeleton was prepared as above, ®xed with
formaldehyde and processed for double immunofluorescent staining of PEBP2b/CBFb and F-actin. The primary
antibody was the anity puri®ed anti-b peptide antibody
(100-fold-diluted) and the secondary antibody was a
mixture of FITC-conjugated goat antiserum against-chick
IgY (100-fold-diluted, Bethyl) and 0.2 unit/ml of RITCconjugated phalloidin (Molecular Probes, Inc.). To label
vinculin, 100-fold-diluted, mouse monoclonal antibody
against vinculin (Biohit Oy) and 20-fold-diluted, RITCconjugated goat antiserum against mouse IgG (Organon
Teknika Corp.) were used. Incubation of the samples with
the respective antibodies was for 30 min at 378C and the
samples were rinsed in PBS after each antibody incubation.
Coverslips were mounted on glass slides with bu€ered
glycerol medium and the cells were viewed with a confocal
laser scanning microscope (LSM410, Zeiss).
EMSA
Preparation of the cytoskeleton
REF52 cells grown in 10 cm-diameter dishes at 1610 cells
were rinsed once with a microtuble stabilizing bu€er (MSB;
0.1 M piperazine-N,N'-bis [2-ethanesulfonic acid], pH 6.9,
2 M glycerol, 5 mM MgCl2 and 2 mM EGTA). The cells
were then covered gently with 500 ml of 0.1% (vol/vol) NP40 in MSB and the solution was quickly recovered and
taken to be the soluble fraction. The remaining material
was scraped into a cold solution of 10% (wt/vol)
trichloroacetic acid. The pellet recovered by centrifugation
was dissolved in 210 ml of a bu€er containing 9 M urea,
2% (vol/vol) Triton X-100, 1% (wt/vol) dithiothreitol, 2%
(wt/vol) lithium dodecylsulfate and 50 mM Tris-HCl,
pH 7.5 and taken to be the cytoskeleton fraction. Protein
concentrations were 0.39 mg/ml for the soluble and 0.94 mg/
ml for the cytoskeleton fraction, respectively. Therefore, the
total yields from 16106 cells were 195 mg and 197 mg
protein for each fraction. Twenty mg of the protein from
each fraction which corresponded to the equivalent cell
number (16105) was subjected to an immunoblot analysis.
For immunocytochemical staining, REF52 cells grown on
coverslips were treated with 0.1% (vol/vol) NP-40 in MSB
as described above, ®xed with 3.7% (wt/vol) formaldehyde
in PBS for 10 min and processed for immunostaining.
6
Whole-cell extracts from 16106 of NIH3T3 cells in 10 cmdiameter dishes were prepared by the freeze-thaw method
as previously described (Lu et al., 1995). The cell pellets
were suspended in 1 ml of a bu€er containing 20 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES]KOH, pH 7.9, 400 mM NaCl, 25% (vol/vol) glycerol, 1 mM
EDTA, 2.5 mM dithiothreitol and 1 mM PMSF, frozen and
thawed three times. The supernatant after centrifugation at
13000 r.p.m. for 10 min was taken to be the extract. The
extracts containing 2 or 4 mg of proteins was incubated in
12 ml of a bu€er containing 20 mM HEPES-KOH, pH 7.6,
4% (wt/vol) Ficoll, 50 mM KCl, 1.5 mg of poly (dI±dC),
2 mM EDTA, 1 mM dithiothreitol and the DNA probe
(1 ng, 2.06105 c.p.m./pmole). The incubation was continued at room temperature for 30 min. Samples were
analysed on 6% (wt/vol) nondenaturing polyacrylamide
(60:1, acrylamide:bisacrylamide) gels. The DNA probe
used was the polyomavirus enhancer sequence (5'GATCCAACTGACCGCAGCTG-3') end labeled with
[32P] by the Klenow enzyme. The underlined sequence
represents a PEBP2 binding site. A mutated version of the
DNA probe harbored the sequence, 5'-GATCCAACTGAACGAAGCTG-3', where the underlined residues
represent the mutated locations.
Western blot (immunoblot) analysis
The protein extract was precipitated by adding trichloroacetic acid to 10% (wt/vol) and the pellet was dissolved in
an SDS ± PAGE sample bu€er. The proteins were separated
by electrophoresis on a 0.1% (wt/vol) SDS ± 12% (wt/vol)
polyacrylamide gel. The transfer of the proteins from the
gel onto the ®lter, the blocking reaction, incubation of the
®lter with the appropriate antibodies and visualization of
the immunocomplexes using ABC kit and ECL detection
reagents were as described previously (Chiba et al., 1997).
Acknowledgements
We thank Ms I Imamura for secretarial assistance. This
investigation was supported in part by research grants from
the Ministry of Education, Science and Culture of Japan,
Proposal-Based Advanced Industrial Technology R&D
Program, Takeda Science Foundation, The Ryoichi Naito
Foundation for Medical Research, The Mochida Memorial
Foundation for Medical and Pharmaceutical Research,
Uehara Memorial Foundation and The Naito Foundation.
References
Aberle H, Butz S, Stappert J, Weissig H, Kemler R and
Hoschuetzky H. (1994). J. Cell Sci., 107, 3655 ± 3663.
Bae S-C, Ogawa E, Maruyama M, Oka H, Satake M,
Shigesada K, Jenkins NA, Gilbert DJ, Copeland NG and
Ito Y. (1994). Mol. Cell. Biol., 14, 3242 ± 3252.
Baeuerle PA and Baltimore D. (1988). Science, 242, 540 ± 546.
Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D,
Grosschedl R and Birchmeier W. (1996). Nature, 382,
638 ± 642.
Affinity of PEBP2b/CBFb with the cytoskeleton
Y Tanaka et al
Burridge K, Fath K, Kelly T, Nuckolls G and Turner C.
(1988). Annu. Rev. Cell. Biol., 4, 487 ± 525.
Chen C and Okayama H. (1987). Mol. Cell. Biol., 7, 2745 ±
2752.
Chiba N, Watanabe T, Nomura S, Tanaka Y, Minowa M,
Niki M, Kanamaru R and Satake M. (1997). Oncogene,
14, 2543 ± 2552.
Erickson P, Gao J, Chang KS, Look T, Whisenant E,
Raimondi S, Lasher R, Trujillo J, Rowley J and Drabkin
H. (1992). Blood, 80, 1825 ± 1831.
Geiger B. (1979). Cell., 18, 193 ± 205.
Geiger B. (1989). Curr. Opin. Cell Biol., 1, 103 ± 109.
Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward
DC, Bray-Ward P, Morgan E, Raimondi SC, Rowley JD
and Gilliland DG. (1995). Proc. Natl. Acad. Sci. USA, 92,
4917 ± 4921.
Huber O, Korn R, McLaughlin J, Ohsugi M, Herrmann BG
and Kemler R. (1996). Mech. Dev., 59, 3 ± 10.
HuÈlsken J, Birchmeier W and Behrens J. (1994). J. Cell Biol.,
127, 2061 ± 2069.
Kagoshima H, Shigesada K, Satake M, Ito Y, Miyoshi H,
Ohki M, Pepling M, and Gergen P. (1993). Trends in
Genetics, 9, 338 ± 341.
Kamachi Y, Ogawa E, Asano M, Ishida S, Murakami Y,
Satake M, Ito Y and Shigesada K. (1990). J. Virol., 64,
4808 ± 4819.
Liu P, Tarle SA, Hajra A, Claxton DF, Marlton P,
Freedman M, Siciliano MJ and Collins FS. (1993).
Science, 261, 1041 ± 1044.
Lu J, Maruyama M, Satake M, Bae SC, Ogawa E,
Kagoshima H, Shigesada K and Ito Y. (1995). Mol. Cell.
Biol., 15, 1651 ± 1661.
McCrea PD, Turck CW and Gumbiner B. (1991). Science,
254, 1359 ± 1361.
Meyers S, Downing JR and Hiebert SW. (1993). Mol. Cell.
Biol., 13, 6336 ± 6345.
Mitani K, Ogawa S, Tanaka T, Miyoshi H, Kurokawa M,
Mano H, Yazaki Y, Ohki M and Hirai H. (1994). EMBO
J., 13, 504 ± 510.
Miyoshi H, Shimizu K, Kozu T, Maseki N, Kaneko Y and
Ohki M. (1991). Proc. Natl. Acad. Sci. USA, 88, 10431 ±
10434.
Nagafuchi A, Ishihara S and Tsukita S. (1994). J. Cell Biol.,
127, 235 ± 245.
Niki M, Okada H, Takano H, Kuno J, Tani K, Hibino H,
Asano S, Ito Y, Satake M and Noda T. (1997). Proc. Natl.
Acad. Sci. USA, in press.
Nucifora G, Begy CR, Erickson P, Drabkin HA and Rowley
JD. (1993). Proc. Natl. Acad. Sci. USA, 90, 7784 ± 7788.
Nucifora G and Rowley JD. (1995). Blood, 86, 1 ± 14.
Ogawa E, Inuzuka M, Maruyama M, Satake M, NaitoFujimoto M, Ito Y and Shigesada K. (1993a). Virology,
194, 314 ± 331.
Ogawa E, Maruyama M, Kagoshima H, Inuzuka M, Lu J,
Satake M, Shigesada K and Ito Y. (1993b). Proc. Natl.
Acad. Sci. USA, 90, 6859 ± 6863.
Okuda T, van Deursen J, Hiebert SW, Grosveld G and
Downing JR. (1996). Cell, 84, 321 ± 330.
Palombella VJ, Rando OJ, Goldberg AL and Maniatis T.
(1994). Cell, 78, 773 ± 785.
Pratt WB. (1993). J. Biol. Chem., 268, 21455 ± 21458.
Romana SP, Mauchau€e M, Le Coniat M, Chumakov I, Le
Paslier D, Berger R and Bernard OA. (1995). Blood, 85,
3662 ± 3670.
Sakakura C, Yamaguchi-Iwai Y, Satake M, Bae SC,
Takahashi A, Ogawa E, Hagiwara A, Takahashi T,
Murakami A, Makino K, Nakagawa T, Kamada N and
Ito Y. (1994). Proc. Natl. Acad. Sci. USA, 91, 11723 ±
11727.
Salgia R, Brunkhorst B, Pisick E, Li JL, Lo SH, Chen LB and
Grin JD. (1995). Oncogene, 11, 1149 ± 1155.
Sasaki K, Yagi H, Bronson RT, Tominaga K, Matsunashi T,
Deguchi K, Tani Y, Kishimoto T and Komori T. (1996).
Proc. Natl. Acad. Sci. USA, 93, 12359 ± 12363.
Shurtle€ SA, Buijs A, Behm FG, Rubnitz JE, Raimondi SC,
Hancock ML, Chan GC-F, Pui C-H, Grosveld G and
Downing JR. (1995). Leukemia, 9, 1985 ± 1989.
Wang S and Speck NA. (1992). Mol. Cell. Biol., 12, 89 ± 102.
Wang Q, Stacy T, Binder M, Marin-Padilla M, Sharpe AH
and Speck NA. (1996a). Proc. Natl. Acad. Sci. USA, 93,
3444 ± 3449.
Wang Q, Stacy T, Miller JD, Lewis AF, Gu T-L, Huang X,
Bushweller JH, Bories J-C, Alt FW, Ryan G, Liu PP,
Wynshaw-Boris A, Binder M, Marin-Padilla M, Sharpe
AH and Speck NA. (1996b). Cell, 87, 697 ± 708.
Wang S, Wang Q, Crute BE, Melnikova IN, Keller SR and
Speck NA. (1993). Mol. Cell. Biol., 13, 3324 ± 3339.
683