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J Biol Chem, Vol. 274, Issue 52, 37070-37078, December 24, 1999
From the HSP Research Institute, Kyoto Research Park, 17 Chudoji
Minami-machi, Shimogyo-ku, Kyoto 600-8813, Japan
The chaperonin containing t-complex
polypeptide 1 (CCT) is a heterooligomeric molecular chaperone assisting
in the folding of actin, tubulin, and other cytosolic proteins. The
expression levels of CCT subunits varied among seven mouse cell lines
tested but showed a close correlation with growth rate. Both the CCT protein and mRNA levels in the human promyelolytic cell HL60
decreased concomitant with growth arrest during differentiation. More
rapid decrease in CCT level occurred when the mouse interleukin
(IL)-3-dependent myeloid DA3 cells were starved for IL-3.
Readdition of IL-3 caused rapid resumption of CCT synthesis during
synchronous growth: the maximum CCT protein and mRNA levels were
observed at G1/S transition through early S phase. The
turnover rate of CCT was nearly constant regardless of growth. Gel
filtration and immunoprecipitation analyses indicated that CCT in
vivo is associated with tubulin at early S phase, but not at
G0/G1 phase. These results demonstrated that CCT expression is strongly up-regulated during cell growth especially from G1/S transition to early S phase and is primarily
controlled at the mRNA level. CCT appears to play important roles
for cell growth by assisting in the folding of tubulin and other proteins.
Many proteins synthesized in the cell require assistance from
molecular chaperones to attain functional conformations (reviewed in
Refs. 1-3). The chaperonin containing t-complex polypeptide 1 (CCT),1 also called TRiC or
c-cpn, is known to mediate folding of proteins in the cytosol (reviewed
in Refs. 4-6). CCT is a member of the chaperonin family that includes
mitochondrial Hsp60, bacterial GroEL, plastid ribulose-bisphosphate
carboxylase/oxygenase subunit-binding protein, and archea group II
chaperonins. CCT shows a double torus-like structure with 8-fold
rotational symmetry (7, 8) assembled from 16 subunits. In mammalian
somatic cells, CCT is composed of eight different subunits of
approximately 60 kDa: CCT is known to assist in the folding of actin and tubulin in the
presence of ATP in vitro (16-21) and to bind newly
synthesized actin and tubulin in vivo (22-24). In budding
yeast, null mutations of CCT subunit genes are lethal, and
temperature-sensitive mutants of individual CCT subunits are affected
in assembly of tubulin and/or actin (reviewed in Ref. 25). Several
other proteins were also reported as possible substrates of CCT.
In vitro, Despite the essential roles of CCT in protein folding, little is known
about the regulation of CCT expression. Here, we report that the CCT
expression is closely correlated with cell growth and is markedly
enhanced at early S phase of the cell cycle in mouse and human cultured
cells. The CCT up-regulated around early S phase is associated with
tubulin in vivo. From these observations, we discuss the
role of CCT in cell growth and cell cycle progression.
Mouse Cultured Cells and Synchronization--
The interleukin
(IL)-3-dependent myeloid cell line DA3 (33),
IL-6-dependent B-cell hybridoma 7TD1 (34), mammary
carcinoma FM3A, lymphoma L5187Y, and myeloma WEHI-3 were obtained from
Riken Cell Bank (Tsukuba, Japan). FM3A, L5187Y, and WEHI-3 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS).
DA3 cells were routinely maintained in 10% FBS/RPMI 1640 supplemented
with 50 units/ml mouse IL-3 (Genzyme, Cambridge, MA) or 9:1 mixture of
10% FBS/RPMI 1640 and WEHI-3 cell-conditioned medium (as IL-3 source).
7TD1 cells were cultured in 10% FBS/RPMI 1640 containing mouse IL-6
(25 units/ml, Genzyme). Lewis lung carcinoma LLC1, fibroblast L929, and
macrophage-like J774A.1 cells were obtained from American Type Culture
Collection (ATCC; Rockville, MD) and cultured in Dulbecco's modified
Eagle's medium containing 10% FBS.
For synchronization, DA3 cells were arrested at
G0/G1 phase by starvation for IL-3 for 16 h in 10% FBS/RPMI 1640, and 7TD1 cells were arrested by starvation for
IL-6 for 20 h. Synchronized cell growth was induced by addition of
these growth factors.
Differentiation of Human HL60 Cells--
Promyelocytic leukemia
HL60 cells (obtained from ATCC) were routinely maintained in 10%
FBS/RPMI 1640. Differentiation of HL60 cells was induced by addition of
1.4% dimethyl sulfoxide (Me2SO), 1 µM
retinoic acid (RA), 1 mM sodium butyrate, or 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA).
Directions and progression of differentiation were monitored by
analyzing morphological changes, expression of cell-surface CD11b
antigen, and induction of two different cytosolic esterase activities
using [3H]Thymidine Incorporation Assay of Growing
Cells--
Cultured cells were inoculated at a concentration of 1 × 105 cells/ml in appropriate media produced by replacing
normal FBS with dialyzed FBS (cutoff size of 2000), and cultured for
8 h. After addition of [3H]thymidine (37 kBq/ml; NEN
Life Science Products) to the media, cells were further cultured for
1 h and then lysed in 0.5 N NaOH. After neutralization
of the lysate with HCl, proteins were precipitated in 10%
trichloroacetic acid. Following three washes in 5% trichloroacetic acid, radioactivity in the precipitate was measured using a liquid scintillation counter.
Analysis of Cell Cycle by DNA Content--
Cells were stained
with propidium iodide (PI) using a CycleTEST PLUS DNA reagent kit
(Becton-Dickinson) and analyzed by the FACSCalibur flow cytometry system.
Antibodies--
Mouse monoclonal antibodies to actin (C4;
Chemicon, Temecula, CA), Western Blotting--
Cells were lysed in 40 mM
Hepes-NaOH buffer (pH 7.5) containing 1% Triton X-100, 5% glycerol, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride
on ice, and the lysate was centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was recovered and protein concentration was determined by the method of Bradford (38), using
bovine serum albumin as a standard. The extracted proteins were
separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 7.5 or
10% polyacrylamide gels (39), and blotted onto polyvinylidene
difluoride filters according to the method of Towbin et al.
(40) using Mini-PROTEAN II equipment (Bio-Rad). After blocking with 5%
(w/v) skim milk in phosphate-buffered saline containing 0.05% Tween
20, the filters were incubated with appropriate first antibody and then
with alkaline phosphatase-conjugated goat antibody against rabbit, rat,
or mouse immunoglobulins (BIOSOURCE International,
Camarillo, CA) in phosphate-buffered saline/Tween 20. The specific
binding of the antibodies was visualized by developing with tetrazolium
bromochloroindolyl phosphate and nitro blue tetrazolium in 50 mM sodium carbonate buffer (pH 9.8) containing 1 mM MgCl2. The resulting bands were scanned with
a flatbed scanner (JX-330; Sharp, Mahwah, NJ) and analyzed using the
public domain NIH Image program (U. S. National Institutes of
Health, Bethesda, MD).
Northern Blotting--
Denhardt's reagent, 20 × SSPE
(1 × SSPE: 150 mM NaCl, 10 mM sodium
phosphate buffer, pH 7.4, 1 mM EDTA), 20 × SSC
(1 × SSC: 150 mM NaCl, 15 mM trisodium
citrate), deionized formamide, and salmon sperm DNA solution were
prepared according to Sambrook et al. (41). Total RNA was
extracted from cultured cells using SV Total RNA Isolation System
(Promega, Madison, WI) according to the manufacturer's instructions.
Total RNA (5 µg/lane) was electrophoresed on 2.2 M
formaldehyde, 1% agarose gels and transferred onto nylon filters.
These filters were hybridized with 32P-labeled human or
mouse Ccta, Cctb, Metabolic Labeling of Proteins and
Immunoprecipitation--
Cells were cultured in methionine (Met)- and
cysteine (Cys)-free RPMI 1640 medium containing 10% dialyzed FBS and
[35S]Met/[35S]Cys mixture (NEN Life Science
Products Inc.), at 1.5 MBq/ml for 15 min, or at 7.4 MBq/ml for 4 h, depending on purpose. The labeled proteins in these cultured cells
were chased in medium containing 10 mM nonradiolabeled Met
and Cys for appropriate periods and extracted using RIPA buffer (1%
Nonidet P-40, 0.1% sodium deoxycholate, 100 mM NaCl, 4 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
0.2 milliunits/ml aprotinin, 50 mM HEPES-NaOH, pH 7.5). After removing proteins that bind to protein G-Sepharose by absorption (Amersham Pharmacia Biotech), aliquots (200 µl) of the supernatant was mixed with 5 µg of anti-CCT Gel Filtration Chromatography--
Cellular proteins were
extracted as described above (see "Western Blotting" section). A
Superose 6 column (1 × 30 cm) (Amersham Pharmacia Biotech) was
equilibrated with a buffer consisting of 40 mM Hepes-NaOH
(pH 7.2), 5 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. The extracted proteins were applied to the column at a
flow rate of 0.5 ml/min with an FPLC system (Amersham Pharmacia Biotech). Protein concentration was monitored by absorbance at 280 nm
and fractions (0.5 ml each) of the eluate were collected. Aliquots of
the fractions were analyzed by Western blotting.
Growth-dependent Expression of CCT in Mouse Cultured
Cells--
Previously, we found that CCT subunits are highly expressed
in mouse tissues containing rapidly propagating cells such as the
testis and spleen (13), suggesting that CCT expression may be
correlated with cell growth. To study this correlation further, we
analyzed CCT expression levels in several lines of mouse cultured cells
that exhibit widely different growth rates. Cellular proteins were
extracted from these cells, and expression levels of the eight
different CCT subunits were determined by Western blotting with
specific antibodies to individual subunits (Fig.
1). The mouse cell lines used in this
experiment were: FM3A (mammary carcinoma), DA3 (myeloid cells), L5187Y
(lymphoma), 7TD1 (B-cell hybridoma), J774A.1 (macrophage-like cells),
LLC1 (lung carcinoma), and L929 (fibroblasts). Under the culture
conditions used, FM3A, 7TD1, DA3, and L5187Y cells grew in suspension,
whereas LLC1, L929, and J774A.1 grew as adherent cells. The CCT
expression levels determined by Western blotting (Fig. 1A)
varied widely among these cells, and were in the following order from
lower to higher: J774A.1 < L929 < LLC1 < L5187Y < DA3 < 7TD1 < FM3A. To examine the correlation between
CCT levels and growth rates, [3H]thymidine incorporation
into DNA was determined with these cells. The radioactivity
incorporated was correlated well with the levels of CCT subunit
expression in these cells (Fig. 1B), with correlation coefficients of 0.89 to 0.97 (Fig. 1D). We also analyzed the
proportion of S phase cells in the cultures by flow cytometry after DNA
staining with propidium iodide and found a reasonable correlation
between % S phase cells and CCT content (data not shown). Since the
folding of tubulin and actin is known to be assisted by CCT (16-18,
21-24), the amounts of these proteins expressed in these cells were
determined. In contrast to CCT, the amounts of three known substrates
of CCT, actin,
Although the CCT expression levels varied among the cell lines, the
expression patterns of subunits were very similar among the eight
subunit species (Fig. 1, A and B). To examine
whether these subunits formed complexes, gel filtration analysis of
cell extracts prepared from J774A.1, L929, DA3, and FM3A was carried out. More than 99% of CCT subunits (each approximately 60 kDa) were
eluted as symmetrical peaks at about 700 kDa as judging from Western
blot analysis of eluates, for any of the four cell lines used (data not
shown). These results indicate that most CCT subunits in
vivo exist as a large complex regardless of cell growth. CCT complexes in growing cells are likely to be formed with almost identical subunit composition, although it can be changed in special circumstances such as differentiated neurites (42). The constant ratio
of subunits is consistent with the fixed arrangement of different CCT
subunits in the complex as proposed by Liou et al. (14), and
implies that there is a tight control mechanism for adjusting the
expression levels of different CCT subunits in growing mammalian cells.
Decrease in CCT Expression Level Concomitant with Growth Arrest
during Human HL60 Cell Differentiation--
Next, we analyzed CCT
expression levels in cells undergoing growth arrest during
differentiation. We used HL60 (35), a multipotent promyelolytic cell
that can differentiate into different types of cells in response to a
variety of chemical reagents: Me2SO and RA induce
granulocytic differentiation, whereas sodium butyrate and TPA induce
monocytic and macrophage-like differentiation, respectively. The
differentiation induced by these chemicals was confirmed by
morphological changes, expression of cell-surface CD11b antigen, and
induction of two esterases as reported previously (35). In the course
of differentiation induced by Me2SO, sodium butyrate, or
TPA treatment, cell numbers doubled during the initial 24 h but
showed little increase thereafter (Fig.
2A). Cell proliferation was
arrested after 2 to 3 days upon RA treatment. Analysis of [3H]thymidine incorporation also confirmed the decrease
of DNA synthesis during differentiation (Fig. 2B). In
addition, flow cytometry analysis of cells stained by the TUNEL method
revealed that only a small fraction of the cells (less than 10%) were
apoptotic during differentiation induced by any of the above chemicals
(data not shown).
Under the conditions described above, the levels of two CCT subunits
(
In contrast to the changes in CCT levels, no significant changes were
observed in Turnover of CCT in Cultured Cells--
Since the level of CCT
showed marked changes concomitant with cell growth as described above,
turnover of CCT may also play important roles in regulating the amounts
of CCT in the cell. For this experiment, we employed mouse DA3 cells
which requires IL-3 for growth (33). When DA3 cells that had been grown
with IL-3 were cultured in the absence of IL-3, the amounts of all CCT
subunits decreased rapidly (Fig. 4).
After IL-3 starvation for 16 h, the amounts of CCT subunits were
reduced to approximately half of the levels in normally growing cells
(Fig. 4B) although the rate of decrease is not completely
identical between subunits (turnover rates may be slightly different).
Even after 32 h of starvation, readdition of IL-3 restored growth
(data not shown) concomitant with coordinated synthesis of CCT subunits
(Fig. 4A). A similar phenomenon was observed using another
growth factor-dependent cell line, 7TD1 (data not shown),
which requires IL-6 for growth (34). These results suggested that CCT
subunits are rapidly degraded during starvation leading to growth
arrest. The amount of
Half-lives of CCT in growing and arrested cells were determined by
pulse-chase experiments. DA3 cells were labeled with
[35S]Met and [35S]Cys for 4 h under
normal growth conditions, and excess unlabeled Met and Cys were added
in the presence or absence of IL-3. The labeled CCT complex was
analyzed by immunoprecipitation using anti-CCT
In the course of these experiments, a 70-kDa protein co-precipitated
with the CCT complex was observed (indicated by asterisks in
Fig. 5). Since HSP70 and/or HSC70 were previously reported to be
co-purified with CCT complex (11, 27, 45), we tested the precipitates
by Western blotting using antibodies specific to either HSP70 or HSC70.
Indeed, the 70-kDa band was recognized by anti-HSC70 (but not by
anti-HSP70) antibody (data not shown). It should be noted that the
HSC70 associated with CCT showed a turnover rate similar to CCT. These
results probably indicate that HSP70 family proteins (e.g.
HSC70 under nonstress conditions) cooperate with CCT in assisting the
folding of cytosolic proteins in vivo, as is the case
in vitro (19, 26, 46).
Cell Cycle-dependent Expression of CCT
Subunits--
To study the correlation between CCT expression and cell
growth in detail, DA3 cells were synchronized and the expression of CCT
was analyzed during the cell cycle. Upon IL-3 starvation for 16 h,
cell growth was arrested uniformly at G0/G1
phase as monitored by flow cytometry with propidium iodide staining of DNA (0 h in Fig. 6A). These
cells showed no significant apoptotic cell death during starvation
(data not shown). After readdition of IL-3, DNA contents changed in a
well synchronized manner (Fig. 6A) in accordance with the
phases of the cell cycle as confirmed by Western blot analysis of
cyclins (Fig. 6, D and G). The cells entered
G1/S transition state at 9 h, S phase at 12 h,
and G2/M phases at 15-18 h after readdition of IL-3. The S
phase of the second cycle started at approximately 24 h although
in a less synchronized manner.
The levels of CCT subunits in the synchronized cells were determined by
Western blotting (Fig. 6, B and E). CCT subunits
were increased by severalfold relative to the initial low level
(20-50% of normally growing cells): it started to increase at 6 h after IL-3 addition, and continued to increase until 12 h. After
reaching the peak at 12 h, CCT level began to decrease at 12-15
h. This was followed by a second increase which started at 18-21 h and continued until 24 h. Both the peaks of CCT level observed at 12 and 24 h coincide with early S phase, indicating that the
abundance of CCT subunits changes during the cell cycle with a maximum
level at early S phase. Synthesis rates of CCT subunits during the cell cycle were then analyzed by pulse labeling for 15 min followed by
immunoprecipitation with an antibody against CCT
On immunoprecipitation by anti-CCT Coordinate Expression and Association of Tubulin with CCT--
In
the above experiments with DA3 cells, we observed that the cellular
levels of
To further examine the physiological significance of growth- and cell
cycle-dependent expression of CCT, we analyzed the CCT and
substrate proteins in the synchronized DA3 cells by a combination of
gel filtration chromatography and immunoprecipitation. Proteins extracted from the synchronized cells were applied onto a Superose 6 column, and the eluted fractions were analyzed by Western blotting using monoclonal antibodies against CCT
Since co-fractionation of CCT with a small amount of actin was observed
in fractions at approximately 400 kDa (Fig. 9, A-C), we
tested whether CCT bound actin in these fractions by
immunoprecipitation with anti-actin antibody. However, no CCT was
detected in the precipitates (data not shown) and thus the actin eluted
in these fractions is not bound to CCT. These results suggest that
actin occupies CCT for a period much shorter than tubulin. In agreement with this view, Sternlicht et al. (22) estimated the
half-lives of CCT-tubulin and CCT-actin complexes in Chinese hamster
ovary cells to be 5-10 min and less than 2-3 min, respectively, and Thulasiraman et al. (24) estimated the latter to be 50 s.
It is also notable that the change of CCT subunit abundance during cell
cycle followed a time course similar to that of cyclin E (specific to
G1/S transition and S phase; Fig. 6, E and
G). Cyclin E is known to be involved in G1/S
phase transition (47, 48). Recently, Won et al. (29)
reported that CCT plays an essential role in cyclin E maturation in
yeast and is associated with cyclin E in human cells (29). Although we
analyzed cyclin E using the Superose 6 fractions, little or no
co-elution of cyclin E with CCT was observed in the present system
(data not shown). CCT may interact with cyclin E transiently as with actin.
Growth- and Cell Cycle-dependent Expression of
CCT--
We presented several lines of evidence indicating that the
expression levels of cytosolic chaperonin CCT are closely correlated with growth rates of mouse and human cells: faster growing cells express CCT subunits at higher levels. This applies to a wide variety
of cells derived from different origins and showing various distinctive
characteristics (fibroblasts, carcinoma, lymphoma, myeloma etc; Fig.
1). Such a correlation is likely to apply also during the
differentiation of cells, since the levels of CCT subunits and their
mRNAs decrease with growth arrest during differentiation of the
promyelotic cell line HL60 (Figs. 2 and 3). The same applies to cells
under growth arrest by growth factor starvation (Fig. 4 and similar
results from the IL-6-dependent cell line 7TD1). These
results indicate that CCT expression is down-regulated at G0/G1 phase of the cell cycle.
These observations are consistent with the following findings reported
previously: mouse tissues abundant in growing cells (e.g.
testis, spleen, thymus, and bone marrow) and cultured cells express
much higher levels of CCT subunits and their mRNAs than those poor
in growing cells (e.g. heart, kidney, and lung) (13, 49,
50). Mouse preimplantation embryos begin to synthesize CCT subunits at
an early cleavage stage (51, 52). In post-implantation embryos,
Tcp-1/Ccta mRNA abundantly expressed at early stages decreases with development (49). Levels of CCT subunits decrease in a
subunit-dependent manner during the RA-induced neuronal
differentiation of the mouse embryonal carcinoma P19 cells (42). In
human keratinocyte cell line K14, CCT synthesis is down-regulated by
agents affecting cell growth and differentiation such as TPA,
interferon
However, CCT expression unrelated to cell growth appears to occur under
special circumstances. When tubulin is rapidly synthesized and
assembled, CCT expression is up-regulated even after growth arrest
(e.g. during spermatogenesis (54) and neurite development (31) of mammals, and cilia generation of Tetrahymena (55, 56)). Apart from such cases, the primary cause for promoting CCT
expression seems to be continuous cell growth.
Since degradation rates of CCT are rapid (t1/2 = 6.0-7.5 h) and do not seem to vary greatly among different cell types
(Fig. 5), CCT subunit levels in vivo can decrease rapidly
once their synthesis is stopped (Fig. 4). It is tempting to speculate
that such instability represents part of the mechanism for regulating
CCT levels. Excess CCT should be removed from the cytosol when cell
growth is arrested because they can be deleterious to the cell when
substrate proteins are not synthesized. In this connection, CCT
expression levels vary by more than 100-fold between the mouse testis
and heart (13).
The growth-dependent CCT expression can be explained by
preferential CCT synthesis during the period from G1/S
transition to early S phase. The CCT levels reach a maximum at this
time in DA3 cells (Fig. 6), whereas the degradation rate of CCT is almost constant in these cells regardless of growth rate (Fig. 5).
Maximum synthesis of CCT subunits occurs at almost the same time as
maximum CCT level (Fig. 7), and is controlled at the mRNA level
(Fig. 8). It is clear that the CCT expression is up-regulated not only
by release from G0 or quiescent phase because up-regulation of CCT around early S phase also occurs in the second round of cell
cycle (Figs. 6-8) and slowly growing cells which are not at G0 phase express less abundant CCT than rapidly growing
cells (Fig. 1). These observations indicate that CCT subunit expression levels are tightly controlled during the cell cycle and this control is
primarily exerted at the mRNA level.
In apparent contradiction to our results, Dittmar et al.
(57) recently reported G2/M phase-specific expression of
CCT Possible Role of CCT in Cell Growth and Cell Cycle
Progression--
It is well known that CCT can assist the folding of
actin and tubulin in vitro (16-18, 20) and that CCT binds
newly synthesized actin and tubulin in vivo (22-24). In the
present study, coregulation of CCT and tubulin was often observed
(Figs. 2-4 and 8) although it is not always (Fig. 1). Since a fraction
of tubulin was found to associate with the CCT complex in early S phase
cells, but not in G0/G1-arrested cells (Fig.
9), one of the CCT role for cell growth seems to be assisting in the
folding of tubulin. On the other hand, the amount of actin showed
little change during differentiation or growth factor starvation but
was slightly up-regulated during synchronous recovery (Figs. 2, 4, and
6). Although these results do not exclude the requirement of CCT for
actin folding, actin may require less abundant CCT than tubulin to fold
the same number of molecules in vivo since the actin
synthesized in vivo is known to occupy CCT more transiently
than tubulin (22, 24) and the actin bound to CCT was below the level of
detection in the present experiments (Fig. 9).
Although the seven kinds of cells used in this study show various
growth rates and contain different levels of tubulin and actin under
normal growth conditions, the expression levels were not significantly
correlated with growth rate (Fig. 1). These results indicate that the
growth-dependent up-regulation of CCT does not always occur
in cells abundant in tubulin or actin, and thus suggest that the
contribution of the abundant CCT in rapidly growing cells is not
restricted to the folding of tubulin and actin. Many other targets of
CCT-assisted folding have been suggested:
One strong candidate of such proteins is cyclin E, because this protein
is known to be a G1 cyclin involved in G1/S
phase transition (47, 48) in association with the
cyclin-dependent kinase Cdk2 (58). CCT is essential for
de novo synthesis of cyclin E in yeast, and cyclin E becomes
associated with CCT before interacting with Cdk2 in human cells (29).
Thus, CCT abundance from G1/S transition to early S phase
may contribute to effective production of active cyclin E. In support
of this idea, the time courses of CCT and cyclin E expression during
synchronous culture of DA3 cells are very similar to each other (Fig.
6), although we could not detect the binding of CCT to cyclin E
probably due to transient association of these proteins, as described above.
We thank Prof. K. R. Willison for
providing antibodies against CCT subunits. We also thank M. Nakayama
and S. Takahara for technical assistance.
*
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.
2
K. R. Willison and G. Hynes, unpublished data.
The abbreviations used are:
CCT, chaperonin
containing t-complex polypeptide 1;
IL, interleukin, FBS,
fetal bovine serum;
Me2SO, dimethyl sulfoxide;
RA, retinoic
acid;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
PAGE, polyacrylamide gel electrophoresis;
HSP70, 70-kDa heat shock protein;
HSC70, cognate of HSP70;
Ccta and Cctb, genes
encoding
Cytosolic Chaperonin Is Up-regulated during Cell Growth
PREFERENTIAL EXPRESSION AND BINDING TO TUBULIN AT
G1/S TRANSITION THROUGH EARLY S PHASE*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 
, 
, 
, 
, 
-1, 
, and 
(reviewed in Ref. 4). In testis germ cells, another subunit, -2,
is also contained in the complex (9). These subunits are encoded by
independent genes and show approximately 30% identity at the amino
acid sequence level (9-13). A fixed arrangement (14) and hierarchy in
ATP binding activity (15) of these subunits in the torus-like complex
have been proposed.
-transducin (20) and firefly luciferase (19,
26) can be folded with the assistance of CCT. Unfolded cofilin (27),
the 22-kDa peroxisomal membrane protein (28), and cyclin E (29) are
also able to bind CCT. In addition, CCT may be involved in vesicular
trafficking (30), neurofilament formation during neurite development
(31), and a Ras-mediated signal transduction pathway in fungi (32).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-naphthyl acetate and naphthol AS-D chloroacetate as
substrates (35). Proportion of cells undergoing apoptotic cell death
was analyzed by two-color flow cytometry using the FACSCalibur system
(Becton-Dickinson, San Jose, CA) after staining by terminal
deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)
method using an Apoptosis Detection System, Fluorescein kit (Promega,
Madison, WI).
-tubulin (3F3; Chemicon), -tubulin
(DM1A; Cedarlane, Ontario, Canada), and the 70-kDa heat shock protein
(HSP70) (C92F3A-5; StressGen, Victoria, BC, Canada), rat monoclonal
antibody to the cognate of HSP70 (HSC70) (1B5; StressGen), and rabbit
polyclonal antibodies against cyclin A, B1, D1, and E (C-19, M-20,
H-295 and M-20, respectively; Santa Cruz Biotech, Santa Cruz, CA) were purchased from the sources shown. Rat anti-CCT monoclonal antibodies 22b and 84a (36) (StressGen) were used to detect mouse and human CCT, respectively. Rabbit polyclonal antibodies against , , , , (designated as BC-1, GC-1, EC-1, TC-1, and THC-2,
respectively (37)), (DM-2),2 and -1 (9)
subunits of CCT were kindly provided by Prof. K. R. Willison.
-actin, and -tubulin
cDNA probes in Northern hybridization buffer (5 × SSPE,
5 × Denhardt's reagent, 1% SDS, 50% formamide, 0.1 mg/ml
salmon sperm DNA) at 42 °C overnight, and then washed in 0.1 × SSC, 0.1% SDS at 68 °C.
antibody (clone 22b) and incubated at 4 °C for 1 h. Protein G-Sepharose beads were then added to the mixture, incubated at 4 °C overnight on a rotator, and washed repeatedly in RIPA buffer. Proteins bound to the resin were extracted by boiling in SDS-PAGE sample buffer and separated by SDS-PAGE using
10% polyacrylamide gels. Radioactivity of bands was counted using a
BAS2000 image analysis system (Fuji Film, Tokyo, Japan).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and -tubulin, showed no significant correlations
with the growth rates (Fig. 1, C and D).
View larger version (28K):
[in a new window]
Fig. 1.
CCT subunit expression is correlated with
cell growth rate. Amounts of CCT subunits in various mouse
cultured cell lines were determined by Western blotting, and growth
rates were monitored by [3H]thymidine incorporation into
DNA. A, proteins extracted from various cells (4 µg/lane)
were separated by SDS-PAGE (7.5% gel), blotted onto polyvinylidene
difluoride filters, and analyzed with antibodies against the eight CCT
subunits, actin, and tubulin. B and C,
correlation between [3H]thymidine incorporation rate and
the relative amounts of CCT subunits (B) or substrates
(C) quantified from the results shown in panel A.
The expression level of each protein in J774A.1 cells was set as 1.00. Eight hours after inoculation of 1 × 105 cells,
[3H]thymidine was added and radioactivity incorporated
into the trichloroacetic acid-insoluble fraction over a period of
1 h was counted (mean of triplicate experiments; S.D. < 5%).
D, correlation coefficient (r2) was
calculated between [3H]thymidine incorporation and amount
of each protein.
View larger version (25K):
[in a new window]
Fig. 2.
Changes in levels of CCT and substrate
proteins expressed in HL60 cells during granulocytic (induced by
Me2SO (DMSO) or RA), monocytic
(by sodium butyrate), and macrophage-like (by TPA) differentiation in
culture. A, growth of HL60 cells determined by cell
number. Cells that did not take up trypan blue were counted.
B, [3H]thymidine incorporation into 1 × 105 cells over a period of 1 h (mean of triplicate
experiments; S.D. < 5%). C-H, expression levels of CCT
(C and E), CCT (D and
F), -tubulin (G), and actin (H).
Proteins extracted from the differentiating cells (2.5 µg/lane) were
analyzed by Western blotting. The results of Western blotting (C,
D, G, and E) and quantified data (E and
F) are shown. The symbols used in panels B, E,
and F are the same as in panel A.
and ) examined by Western blotting decreased during treatment
with the chemicals (Fig. 2, C-F). These results are consistent with the idea that CCT expression is closely correlated with
cell growth rate. The time course of the decrease in expression level
was similar between the two subunits but different among the inducers
utilized. The CCT levels decreased to less than 5% by TPA treatment
for 4 days, but only to 20-50% after 5 days with the other chemicals
(Fig. 2, E and F). The rapid decrease in CCT induced by TPA is probably related to a rapid decrease in DNA synthesis
(Fig. 2B). It is notable that the decrease in CCT subunit levels had already started within 24 h of treatment with all the inducers used, whereas cell number doubled during this period. These
observations suggest that the synthesis of CCT subunits is reduced
prior to growth arrest determined by counting cell numbers. The levels
of CCT mRNAs encoding and subunits also decreased during treatment with all the inducers: the mRNA levels had started to decrease within 12 h of treatment (Fig.
3). Thus, the CCT mRNA levels
decrease before the onset of growth arrest of these cells, suggesting
that cells rapidly reduce transcription of CCT genes prior
to growth arrest. Taken together, these results indicate that CCT
expression is tightly coupled with cell growth and is mainly controlled
at the mRNA level.
View larger version (42K):
[in a new window]
Fig. 3.
Changes in levels of mRNAs encoding CCT
subunits ( and
), -tubulin, and
-actin in HL60 cells during differentiation induced
by Me2SO (DMSO), RA, sodium butyrate, or
TPA. Total RNA was electrophoresed and blotted onto nylon filters.
The blotted filters were hybridized with 32P-labeled human
CCTA, CCTB, -tubulin, and -actin cDNA
probes and washed in 0.1% SDS, 0.1 × SSC at 68 °C.
-tubulin level except for cultures treated with TPA
(Fig. 2G). Upon TPA treatment, HL60 cells can differentiate to macrophage-like cells without cell division or new DNA synthesis (43), accompanied by phosphorylation of tubulin and
microtubule-associated proteins followed by destabilization and
depolymerization of microtubule architecture (44). Since TPA treatment
also leads to rapid reduction of CCT subunit level, tubulin and CCT
appears to be coregulated in these cells. The level of -tubulin
mRNA decreased rapidly in response to all of the inducers tested
(Fig. 3), similarly to CCT subunit mRNA level, suggesting that
transcription of these mRNAs may also be co-regulated. On the other
hand, the amount of actin showed little change during differentiation
induced by any of these chemicals (Fig. 2H) and the actin
mRNA showed little change or slow decrease depending upon the
chemicals used (Fig. 3).
-tubulin was reduced significantly during
starvation but that of actin remained unaffected (Fig. 4).
View larger version (27K):
[in a new window]
Fig. 4.
Changes in CCT subunit levels in DA3 cells
during growth arrest by IL-3 starvation and recovery of growth by
readdition of IL-3. DA3 cells were starved for IL-3 for 32 h,
and then cultured in the presence of IL-3 (50 units/ml) for
12 h. A, at the intervals indicated, cells were
collected, and extracted proteins (2.5 µg/lane) were analyzed by
Western blotting using antibodies against CCT subunits, -tubulin,
and actin. B, bands shown in panel A (except
after IL-3 readdition) were quantified and plotted against time of
starvation.
monoclonal antibody
followed by SDS-PAGE (Fig.
5A). Since eight different
subunits of approximately 60 kDa are contained in the CCT hexadecamer
complex (4), they appear as a cluster of bands at 53-65 kDa by
SDS-PAGE (11, 16, 17, 45). The half-life of labeled CCT in DA3 cells
was estimated to be 6.0 h in the presence of IL-3 and 6.8 h
in the absence of IL-3 (Fig. 5B), indicating that the
turnover rate of CCT is little affected by growth conditions. Another
cell line, FM3A, which grows faster and shows higher CCT contents (Fig.
1), showed a similar CCT half-life (7.5 h) under normal growth
conditions (Fig. 5). These results indicate that CCT undergoes rapid
degradation with nearly constant rates regardless of growth conditions
and probably also of cell type.
View larger version (27K):
[in a new window]
Fig. 5.
Turnover of CCT in cultured cells.
A, exponentially growing DA3 or FM3A cells (2 × 105 cells/ml) were labeled with a mixture of
[35S]Met and [35S]Cys (7.4 MBq/ml) for
4 h, and chased by addition of nonlabeled Met and Cys. CCT was
immunoprecipitated with anti-CCT antibody (clone 22b) and protein
G-Sepharose. The precipitated proteins were separated by SDS-PAGE (10%
gel). Multiple bands for CCT reflect eight different subunits of
various sizes, and asterisks indicate the Hsc70 protein
coprecipitated with CCT. B, radioactivity of CCT was
quantified from the gels shown in panel A.
View larger version (38K):
[in a new window]
Fig. 6.
Changes in CCT subunit levels during
synchronized growth of DA3 cells. Cells were starved for IL-3 for
16 h, and IL-3 was added at time 0. A, DNA histogram.
Cells were stained with PI and analyzed by flow cytometry.
B-G, Western blot analysis of CCT subunits (B),
CCT substrate proteins ( - and -tubulin, and actin; panel
C), and cyclins (D). Cells were collected at the times
indicated and the extracted proteins (2.5, 1, and 10 µg/lane for
B, C and D, respectively) were analyzed with
specific antibodies. Cyclin E migrates as two bands of 55 and 50 kDa
(47). AS, asynchronous culture. Panels E, F, and
G represent quantified data from panels B, C, and
D, respectively.
(Fig.
7). These results indicated that maximum
synthesis occurs at 12 and 21-24 h corresponding to early S phase. The
total protein synthesis determined from trichloroacetic
acid-precipitable radioactivity started much earlier (with the first
peak at 6 h; Fig. 7B) than CCT synthesis, suggesting
that the proteins synthesized at the early stage (0-6 h) are less
dependent on CCT-assisted folding and that some of the proteins highly
expressed around early S phase require abundant CCT for folding. In
agreement with the results of CCT synthesis, the levels of mRNAs
encoding CCT subunits determined by Northern blotting (Fig.
8) started to increase within 3 h
and reached the first peak at 9 h. This was followed by a decrease
at 12-15 h and a second increase at 18-21 h.
View larger version (51K):
[in a new window]
Fig. 7.
Synthesis rates of CCT and total proteins in
the synchronized DA3 cells. A, CCT synthesis was
analyzed by labeling cells with [35S]Met and
[35S]Cys for 15 min followed by lysis and
immunoprecipitation with anti-CCT antibody. Single and
double asterisks indicate Hsc70 and a 50-kDa protein
coprecipitated with CCT, respectively. B, quantified CCT
radioactivity relative to that of asynchronous culture (set as 1.00),
and total protein synthesis rate determined by measuring radioactivity
of trichloroacetic acid-insoluble fractions.
View larger version (68K):
[in a new window]
Fig. 8.
Levels of mRNAs encoding CCT subunits
( and ),
-tubulin, and -actin in
the synchronized DA3 cells. Total RNA samples were analyzed by
Northern blotting using mouse Ccta, Cctb, -tubulin, and
-actin cDNA probes.
antibody, pulse-labeled proteins
of 70 and 50 kDa were coprecipitated with CCT subunits appearing at
53-65 kDa (Fig. 7A). The 70-kDa protein is HSC70 as
described above, whereas the 50-kDa protein is probably tubulin because
their molecular masses are similar, tubulin is known to be copurified
with CCT (22, 37) and association between tubulin and CCT can also be
detected using DA3 cells as described in the next section. Since the
50-kDa protein coprecipitated with CCT gave signals comparable with CCT
by labeling for 15 min (Fig. 7A) but not by labeling for
4 h (Fig. 5A), association between this protein and CCT
is probably transient. This agrees with the previous observation
indicating that the half-life of CCT association with newly synthesized
tubulin in vivo is 5-10 min (22). It should be noted that
the band intensity of the 50-kDa protein changed during cell cycle in
the manner nearly identical to that of CCT.
- and -tubulin decreased during IL-3 starvation and
increased by 2- to 3-fold upon readdition of IL-3, although -tubulin
was more periodic than -tubulin (Fig. 6, C and
F). The level of -tubulin mRNA, which had been
greatly reduced during starvation, increased slowly after IL-3
readdition (Fig. 8). On the other hand, the actin level showed little
change during starvation but slightly up-regulated (25% increase) upon
readdition of IL-3. The small extent of changes may be due to very
large amount of actin pool in the cell because the -actin mRNA
reduced during starvation, recovered very rapidly within 3 h after
IL-3 readdition. These results suggested that the expression of tubulin
and/or actin was partly co-regulated with CCT in DA3 cells
, -tubulin, or actin (Fig.
9). When proteins from the
G0/G1-arrested cells (starved for IL-3 for
16 h) were analyzed, no significant correlations were observed
between the elution peaks of CCT and those of actin or tubulin (Fig.
9A). However, at G1/S transition state (9 h
after IL-3 readdition), a small but significant amount of tubulin was co-eluted with CCT complex around the 670-kDa marker (Fig.
9B). At early S phase (12 h after IL-3 readdition), a much
greater portion of tubulin was co-eluted with CCT (Fig. 9C).
We also determined the amounts of -tubulin in these fractions and
the same results were obtained (data not shown). To confirm the cell
cycle-dependent binding of CCT to tubulin, the CCT peak
fractions around 670 kDa were pooled and immunoprecipitated with
anti--tubulin antibody, and the precipitates were analyzed by
Western blotting with anti-CCT antibody (Fig. 9D). A weak
band of 60 kDa was observed with the fractions from G1/S
transition state (lane 2) and the signals became much
stronger at early S phase (lane 3), in contrast to the
sample from G0/G1 phase that gave no signal
(lane 1). These results clearly indicate that the CCT
complex in vivo associates with tubulin at early S phase,
but not at G0/G1 phase.
View larger version (19K):
[in a new window]
Fig. 9.
Association of tubulin with CCT in DA3 cells
at early S phase. DA3 cells were starved for IL-3 for 16 h,
and IL-3 was added to obtain synchronized cultures. After the
starvation for 16 h (G0/G1 phase;
panels A and E), or after readdition of IL-3 for
9 h (G1/S transition; panels B and
F) or 12 h (early S phase; panels C and
G), cells were collected and lysed. Proteins extracted from
1 × 107 cells were applied onto a Superose 6 column.
A-C, the fractions eluted from the column were collected and
analyzed by Western blotting with antibodies against CCT ,
-tubulin, or actin (fraction numbers 15 to 34, from left
to right). Filters were developed until the same total
intensity of -tubulin was obtained for each panel. D, CCT
peak fractions around 670 kDa shown in panels A (lane
1), B (lane 2), and C (lane
3) were immunopreciptated with anti--tubulin antibody (DM1A)
and analyzed by Western blotting with anti-CCT antibody (TC-1).
E-G, elution profiles of total protein (absorbance at 280 nm) by gel filtration. Numbers indicate elution peaks of
molecular weight standards; bovine thyroglobulin (670,000),
-globulin (158,000) and chicken ovalbumin (44,000).
Vo, void volume; -tub, -tubulin;
act, actin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and okadaic acid (36). Even in an evolutionarily
distant organism, Dictyostelium discoideum, the
Tcp-1/Ccta mRNA level decreases rapidly during
development (53).
(no CCT protein at G1 and S phases) using Ehrlich
ascites tumor cells separated by centrifugal elutriation, although its
mRNA was detectable by Northern blotting throughout the cell cycle.
The cell line used was different from those examined here. However,
this is unlikely to be the reason for the different conclusions since we obtained consistent results using many kinds of cell lines. In
addition, large amounts of signals on CCT can be detected in non-M
phase cells by indirect immununofluorescence of monkey CV-1 (45) and
human HEp-2 (36) cells. Thus, the basis of this contradictory result
remains unclear unless Ehrlich ascites tumor cells are an exceptional case.
-transducin (20) and
firefly luciferase (19, 26) can be folded by assistance of CCT in
vitro. CCT binds unfolded cofilin (27), the 22-kDa peroxisomal
membrane protein (28) and cyclin E (29). Recently, Thulasiraman
et al. (24) detected more than 70 different CCT-bound
polypeptides in vivo by two-dimensional gel electrophoresis
of immunoprecipitated proteins. These observations indicate that CCT
can interact with many proteins other than tubulin and actin. It is
likely that CCT assists in the maturation of proteins up-regulated at
G1/S transition and/or early S phase.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 81-75-315-8656;
Fax: 81-75-315-8659; E-mail: kubota@hsp.co.jp.
ABBREVIATIONS
and subunits of CCT, respectively.
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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 S. Hirayama, Y. Yamazaki, A. Kitamura, Y. Oda, D. Morito, K. Okawa, H. Kimura, D. M. Cyr, H. Kubota, and K. Nagata MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination Mol. Biol. Cell, March 1, 2008; 19(3): 899 - 911. [Abstract] [Full Text] [PDF]
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P. C. Stirling, M. Srayko, K. S. Takhar, A. Pozniakovsky, A. A. Hyman, and M. R. Leroux Functional Interaction between Phosducin-like Protein 2 and Cytosolic Chaperonin Is Essential for Cytoskeletal Protein Function and Cell Cycle Progression Mol. Biol. Cell, June 1, 2007; 18(6): 2336 - 2345. [Abstract] [Full Text] [PDF]
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 M Yoshioka, A Boivin, P Ye, F Labrie, and J St-Amand Effects of dihydrotestosterone on skeletal muscle transcriptome in mice measured by serial analysis of gene expression. J. Mol. Endocrinol., April 1, 2006; 36(2): 247 - 259. [Abstract] [Full Text] [PDF]
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 T. Shimizu, S.-i. Yokota, S. Takahashi, Y. Kunishima, K. Takeyama, N. Masumori, A. Takahashi, M. Matsukawa, N. Itoh, T. Tsukamoto, et al. Membrane-Anchored CD14 Is Important for Induction of Interleukin-8 by Lipopolysaccharide and Peptidoglycan in Uroepithelial Cells Clin. Vaccine Immunol., September 1, 2004; 11(5): 969 - 976. [Abstract] [Full Text] [PDF]
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 S.-i. Yokota, N. Yokosawa, T. Okabayashi, T. Suzutani, S. Miura, K. Jimbow, and N. Fujii Induction of Suppressor of Cytokine Signaling-3 by Herpes Simplex Virus Type 1 Contributes to Inhibition of the Interferon Signaling Pathway J. Virol., June 15, 2004; 78(12): 6282 - 6286. [Abstract] [Full Text] [PDF]
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 C. E. Grossman, Y. Qian, K. Banki, and A. Perl ZNF143 Mediates Basal and Tissue-specific Expression of Human Transaldolase J. Biol. Chem., March 26, 2004; 279(13): 12190 - 12205. [Abstract] [Full Text] [PDF]
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S.-i. Yokota, N. Yokosawa, T. Kubota, T. Okabayashi, S. Arata, and N. Fujii Suppression of Thermotolerance in Mumps Virus-infected Cells Is Caused by Lack of HSP27 Induction Contributed by STAT-1 J. Biol. Chem., October 24, 2003; 278(43): 41654 - 41660. [Abstract] [Full Text] [PDF]
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F. Bai, J. H. Xi, E. F. Wawrousek, T. P. Fleming, and U. P. Andley Hyperproliferation and p53 Status of Lens Epithelial Cells Derived from {alpha}B-crystallin Knockout Mice J. Biol. Chem., September 19, 2003; 278(38): 36876 - 36886. [Abstract] [Full Text] [PDF]
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Y. Yamazaki, H. Kubota, M. Nozaki, and K. Nagata Transcriptional Regulation of the Cytosolic Chaperonin {theta} Subunit Gene, Cctq, by Ets Domain Transcription Factors Elk-1, Sap-1a, and Net in the Absence of Serum Response Factor J. Biol. Chem., August 15, 2003; 278(33): 30642 - 30651. [Abstract] [Full Text] [PDF]
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 J. H. Xi, F. Bai, and U. P. Andley Reduced survival of lens epithelial cells in the {alpha}A-crystallin-knockout mouse J. Cell Sci., March 15, 2003; 116(6): 1073 - 1085. [Abstract] [Full Text] [PDF]
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 A. L. Astier, R. Xu, M. Svoboda, E. Hinds, O. Munoz, R. de Beaumont, C. D. Crean, T. Gabig, and A. S. Freedman Temporal gene expression profile of human precursor B leukemia cells induced by adhesion receptor: identification of pathways regulating B-cell survival Blood, February 1, 2003; 101(3): 1118 - 1127. [Abstract] [Full Text] [PDF]
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H. Kubota, S.-i. Yokota, H. Yanagi, and T. Yura Transcriptional Regulation of the Mouse Cytosolic Chaperonin Subunit Gene Ccta/t-Complex Polypeptide 1 by Selenocysteine tRNA Gene Transcription Activating Factor Family Zinc Finger Proteins J. Biol. Chem., September 8, 2000; 275(37): 28641 - 28648. [Abstract] [Full Text] [PDF]
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