(Received for publication, January 18, 1996; and in revised form, January 30, 1996)
From the
Loss of adhesion leads to cell cycle arrest at the
G/S boundary in normal, adhesion-dependent, mesenchymal
cells. This arrest is accompanied by the inability to produce cyclin A.
Using deletional and mutational analysis of the cyclin A promoter, we
have identified a CCAAT element that mediates the adhesion-dependent
transcriptional activation of cyclin A in late G
phase of
the cell cycle. Specific binding of a novel 40/115-kDa heterodimeric
protein complex, which we have named CBP/cycA, to this CCAAT
element was detectable in growing but not in G
-arrested or
nonadherent normal rat kidney fibroblasts. During G
CBP/cycA appears to be present but sequestered by a
retinoblastoma family member. These results suggest that expression of
cyclin A, which controls cell cycle progression by adhesion at the
G
/S boundary, is regulated by CBP/cycA and the
phosphorylation status of the retinoblastoma protein or a
retinoblastoma-related protein.
Cells of most tissues, with the exception of hematopoietic
cells, require adhesion to a surface in order to grow. Nonadherent
cells fail to proliferate despite the presence of growth
factors(1) . Transformation of cells by
c-Ha-ras(2) , v-src(3) ,
c-sis(4) , or the DNA tumor virus proteins SV40 large
T antigen(5) , adenovirus E1A(6) , and human
papillomavirus E7 (7) results in loss of adhesion dependence.
This in vitro phenotype correlates well with tumorigenicity in vivo(8) . Recently, it has been shown that the
adhesion requirement in mesenchymal cells is likely to reflect a cell
cycle checkpoint in the late G phase of the cell
cycle(9) . Cells arrested by suspension failed to produce
cyclin A(10) . In addition, ectopic expression of cyclin A
enabled the cells to bypass the adhesion requirement, implicating
cyclin A as the major target of cell cycle control by the
anchorage-signaling pathway. The adhesion signal itself appears to be
mediated through adhesion plaques, since modulation of expression of
components of adhesion plaques like integrins(11) ,
vinculin(12) , actinin(13) , or focal adhesion kinase (14, 15) affects the adhesion dependence of cells.
However, little is known about the signal transduction pathway by which
the adhesion signal exerts control of the cell cycle.
In this
report, we have examined the mechanism by which the expression of
cyclin A is regulated by cell adhesion. We show that the
adhesion-dependent transcriptional activation of the cyclin A gene at
the G/S boundary is mediated by a CCAAT element in its
promoter region. This activation is accompanied by binding of a
40/115-kDa heterodimeric protein complex to the CCAAT element. In
addition, we demonstrate that during the G
phase of the
cell cycle this novel trans-acting factor, which we have named
CBP/cycA(
)(CCAAT-Binding Protein for cyclin A gene), seems to be sequestered and thereby
held inactive by a retinoblastoma protein (Rb) family member.
Addition of growth factors to adherent,
G-arrested NRK cells enabled their re-entry into the cell
cycle and resulted in a 20-fold induction of transcription from the
929-bp cyclin A promoter (pWt929; Fig. 1, A and B). However, when cells were plated on polyHEMA-coated dishes
in methylcellulose-containing medium to prevent cell adhesion and
aggregation, only a 2-fold induction of luciferase activity was seen.
This demonstrates that transcription initiated by the cyclin A promoter
is only induced when the cells are attached to a substratum. Deletions
within the cyclin A promoter (Fig. 1A), which included
potential binding sites for the transcription factors E2F (pWt918),
AP1, and E2F (pWt434) or AP1, E2F, and Sp1 (pWt284), did not affect
induction of the cyclin A promoter (Fig. 1, A and B). Similarly, when a 3-bp mutation was introduced into the
remaining E2F site of pWt434 (pMut434), inducibility of the cyclin A
promoter was not affected. The additional deletion of a CCAAT element
and an ATF site (pWt184) decreased the inducibility of luciferase
activity in adherent cells to <1% of pWt929. A small portion of this
decrease resulted from loss of the ATF site
(pWt225)(24, 25, 26) ; however, inducibility
of the cyclin A promoter in adherent cells was still retained after
deletion of the ATF site. Only removal of the CCAAT element located at
-56 to -52 resulted in complete ablation of inducibility.
CCAAT elements have been shown to mediate cell cycle-dependent
transcriptional activation of the human histone H1(27) , heat
shock protein 70 (hsp70)(28) , DNA polymerase
(29) , and thymidine kinase genes (30, 31, 32) at the G
/S boundary.
We therefore introduced a mutation into the CCAAT element of pWt284
(pMut284, Fig. 1A), which completely abolished cyclin A
promoter inducibility (Fig. 1B). Thus, the CCAAT
element is necessary for the adhesion-dependent activation of cyclin A
expression at the G
/S boundary.
Figure 1:
Contribution of potential transcription
factor binding sites to the adhesion-dependent activation of the cyclin
A promoter. A, schematics of the cyclin A promoter region in
plasmids pWt929, pWt918, pWt434, pMut434, pWt284, pMut284, pWt225, and
pWt184, which contain the luciferase gene as a reporter. B,
synchronous, serum-depleted NRK cells were transiently transfected with
the plasmids mentioned above using lipofection. Luciferase activity in
cellular extracts from (i) serum-depleted, G-arrested, (ii)
serum-supplemented, nonadherent, and (iii) serum-supplemented, adherent
NRK cells is presented normalized to G
-arrested NRK cells (n = 3).
To directly demonstrate
an interaction between the CCAAT element of the cyclin A promoter and a
CCAAT-binding protein, EMSA were performed using a 24-bp, CCAAT
element-containing cyclin A promoter fragment as a probe (Fig. 2A). No sequence-specific DNA-protein complexes
were detectable when nuclear extracts from serum-depleted,
G-arrested or serum-supplemented, nonadherent NRK cells
were incubated with the probe. Formation of a sequence-specific
DNA-protein complex was observed when nuclear extracts from
serum-supplemented, adherent NRK cells or actively dividing HeLa cells
grown in suspension were used. Formation of this DNA-protein complex
failed to occur with either nuclear extract when the probe contained a
mutant CCAAT element (Fig. 2B). We named the
DNA-binding protein complex CBP/cycA. UV cross-linking
experiments and a modified Ferguson analysis revealed that CBP/cycA from NRK cells is a 155-kDa heterodimeric protein complex with
subunit sizes of approximately 40 and 115 kDa (Fig. 2C). Identical results were obtained when
CBP/cycA from HeLa cells was used (data not shown). Several
proteins that specifically recognize CCAAT elements have been
described, including CTF/NF-1(33) , CP1 and CP2(34) ,
CBP(35) , NF-Y (36) , C/EBP(37) , and
CBP/tk(32) , among others. Most of these are composed of two
heterologous subunits(27, 31, 34) . Our
finding that CBP/cycA is a heterodimer with subunit sizes of
approximately 40 and 115 kDa is consistent with these reports. However,
to our knowledge, no CCAAT-binding protein subunit of 115 kDa has been
described.
Figure 2:
Formation of a specific complex between
the CCAAT element of the cyclin A promoter and a 40/115-kDa
heterodimeric protein. A, EMSA was performed using a
double-stranded, 24-bp, cyclin A promoter fragment
(5`-CGAGCGCTTTCATTGGTCCATTTC-3` (Wt)) as a probe and nuclear extracts
from (i) serum-depleted, G-arrested, (ii)
serum-supplemented, nonadherent, (iii) serum-supplemented, adherent NRK
cells, and (iv) nuclear extracts from HeLa cells grown in suspension.
The arrow indicates a specific complex formed between Wt probe
and nuclear protein, which we designated CBP/cycA. B,
EMSA using either Wt or a single base mutant, double-stranded, 24-bp,
cyclin A promoter fragment (5`CGAGCGCTTTCATgGGTCCATTTC-3` (Mut)) as a probe and nuclear extracts from
serum-supplemented, adherent NRK cells. C, monomer sizes of
CBP/cycA. A 5-bromo-2`-deoxyuridine-substituted Wt probe was
covalently linked to CBP/cycA by UV-crosslinking (UVX).
As demonstrated, cyclin A transcription is suppressed by
suspension of NRK cells. This can be explained by two mechanisms:
either by an adhesion-responsive repressor element or by failure to
activate transcription through positive regulatory elements at the
G/S boundary. We found no indication of a repressor
element, since none of our reporter construct deletions abolished
transcriptional repression in response to suspension. However, the
transcriptional activity of the cyclin A promoter appears to be
dependent upon binding of CBP/cycA to the CCAAT element. Since
CBP/cycA was not found in nonadherent cells, we conclude that
the transcriptional regulation of cyclin A by adhesion is at least in
part mediated by CBP/cycA. This indicates that the adhesion
signal regulates cell cycle progression prior to the transcriptional
activation of cyclin A at the G
/S boundary. Progression
through the G
phase of the cell cycle is controlled by Rb
and Rb-related proteins (p107, p130). While underphosphorylated during
G
and the first half of G
, Rb becomes
phosphorylated by cyclin D-cyclin-dependent kinase (cdk) 4/cdk6 (38) and cyclin E-cdk2 (39) in the second half of the
G
phase, thereby releasing a number of cellular proteins
including the transcription factor E2F from its bound form to an active
state(40) . Adenovirus E1A (41) , SV40 large T
antigen(42) , and human papillomavirus E7 (43) mimic
these events by binding to hypophosphorylated Rb and displacing
cellular proteins sequestered by Rb. Recently, it has been described
that the transcriptional activation of the hsp70 gene in late
G
by adenovirus E1A is mediated by a CCAAT-binding
protein(28) . In addition, it has been demonstrated that the
Rb-related protein p107 is able to repress the activation of a CCAAT
element-containing herpesvirus thymidine kinase promoter fragment at
the G
/S boundary(44) . This raises the question
whether the adhesion-dependent interaction of CBP/cycA with
the CCAAT element of the cyclin A promoter is regulated by association
with an Rb family member. We therefore preincubated nuclear extracts
from G
-arrested NRK cells with a GST fusion protein that
contained the Rb binding domain of SV40 large T antigen (residues
101-249; GST101-249Tag) in order to release
CBP/cycA. As shown in Fig. 3A, this
pretreatment led to the formation of a DNA-protein complex
indistinguishable from CBP/cycA in EMSA. The same result was
obtained when GST-HPV E7 was used for preincubation (data not shown).
This suggests that in NRK cells CBP/cycA is inactivated during
G
by binding to Rb or an Rb family member. In contrast to
fibroblasts, HeLa cells that are transformed by human papillomavirus 18
are able to grow in suspension. This adhesion-independent growth is
dependent on the expression of human papillomavirus 18 E7(45) .
Our finding that active CBP/cycA is present in HeLa cells
grown in suspension but not in nonadherent NRK cells is consistent with
these data.
Figure 3:
Displacement of CBP/cycA from
binding to an Rb family member. A, displacement of
CBP/cycA by preincubation of nuclear extracts from
G-arrested NRK cells with a GST fusion protein that
contained the Rb binding domain of SV40 large T antigen
(GST101-249Tag). Nuclear extracts from G
-arrested NRK
cells were preincubated with GST101-249Tag for 50 min at 4
°C. Then, an EMSA was performed using a double-stranded, 24-bp,
cyclin A promoter fragment (5`-CGAGCGCTTTCATTGGTCCATTTC-3`) as a probe.
The arrow indicates a DNA-protein complex with the same
electrophoretic mobility as CBP/cycA bound to the probe.
GST101-249Tag was incubated with the probe in the absence of
nuclear extract as a control. B, displacement of CBP/cycA by phosphorylation of nuclear extracts from G
-arrested
NRK cells by cyclin E-cdk2 complexes immunoprecipitated from NRK cells.
Nuclear extracts from G
-arrested NRK cells were
preincubated with immunoprecipitated cyclin E-cdk2 complexes from NRK
cells for 30 min at 37 °C. As a control, cyclin E-cdk2
immunoprecipitates were incubated with the probe in the absence of
nuclear extract.
We have previously been able to show that the kinase
activity associated with cyclin E, which is maximally expressed at the
G/S boundary(20) , is regulated by adhesion and
might therefore constitute an element of the anchorage-signaling
pathway governing the expression of cyclin A. (
)Therefore,
we attempted to release CBP/cycA from its sequestered state by
phosphorylation of Rb and Rb-like proteins with cyclin E-cdk2 kinase.
As shown in Fig. 3B, preincubation of nuclear extracts
from G
-arrested NRK cells by cyclin E-cdk2 complexes
immunoprecipitated from NRK cells resulted in the formation of a
DNA-protein complex indistinguishable from CBP/cycA in EMSA.
To our knowledge, CBP/cycA represents the first CCAAT-binding
protein, which appears to be sequestered by an Rb family member.
In
summary, we demonstrate that activation of cyclin A transcription at
the G/S boundary is dependent upon adhesion of mesenchymal
cells to a surface. The transcriptional activation is mediated by a
novel heterodimeric CCAAT-binding protein that we have named
CBP/cycA. During the G
phase of the cell cycle,
CBP/cycA seems to be sequestered and thereby kept inactive by
Rb or an Rb-related protein. The adhesion signal results in the release
of CBP/cycA from binding to an Rb family member and enables
the induction of cyclin A expression and progression through the
G
/S adhesion checkpoint. Our model predicts that the
control of cell cycle progression by adhesion is exerted through
regulation of the phosphorylation status of Rb family members.