(Received for publication, January 19, 1996; and in revised form, February 16, 1996)
From the
Stimulation of quiescent Balb/c 3T3 fibroblasts into S phase
requires the synergistic action of platelet-derived growth factor
(PDGF) and progression factors found in platelet-poor plasma (PPP).
Traverse of the G/S phase boundary and the initiation of
DNA replication require functional cyclin E-cyclin-dependent kinase
(Cdk) 2 and cyclin A-Cdk2 complexes; however, the mechanisms by which
PDGF and PPP regulate Cdk2 activation are not known. Density-arrested
fibroblasts contain low levels of cyclins E and A, and high levels of
the Cdk inhibitor p27
. Exposure to PDGF, which
stimulates cell cycle entry but not progression through G
,
induces the formation of cyclin D
-Cdk4 complexes that bind
p27
and titrate the pool of Kip1 available to
inhibit Cdk2. In addition, PDGF stimulates a moderate transient
reduction in the abundance of p27
protein.
However, limited expression of cyclin E and cyclin A is observed after
PDGF treatment, and in the absence of PPP, p27 levels are sufficient to
bind and inactivate existing cyclin-Cdk complexes. Although plasma does
not significantly increase the proportion of Kip1 bound to cyclin
D
-Cdk4, stimulation of PDGF-treated cells with plasma does
overcome the threshold inhibition of p27
by
further increasing the expression of cyclins E and A and decreasing the
amount of Kip1 over a prolonged time period. Our results indicate that
the distinct mitogenic activities of PDGF and PPP differentially
influence the activation of cyclin E- and cyclin A-associated kinases
that ultimately regulate entry into S phase.
The growth of nontransformed eukaryotic cells is controlled by
environmental cues which govern the transition from G into
S phase. In mammalian cells, growth regulatory signals from
serum-derived growth factors are integrated during a late G
event called the restriction point(1) . Successful
execution of this event commits cells to another round of DNA
replication, at which time cell cycle progression becomes independent
of extracellular mitogens.
Growth factor-stimulated proliferation is
achieved, at least in part, by a modulation of the cell cycle machinery
consisting of the cylin-dependent kinases (Cdks) ()and their
regulatory cyclin subunits(2, 3) . While a single
kinase, p34
, is sufficient to drive progression
through the major cell cycle regulatory points at the G
/S
and G
/M phase transitions in yeast(4) , multiple
distinct Cdc2-related kinases have been identified in higher
eukaryotes(5, 6, 7) . The involvement of many
of these kinases in cell cycle regulation has yet to be established.
However, Cdk4 becomes active as a retinoblastoma protein kinase during
mid G
(8) , and overexpression of Cdk4 in epithelial
cells reduces the requirement for serum-derived growth factors and
confers resistance to TGF-
-mediated growth inhibition(9) .
Furthermore, Cdk2 rescues the growth-arrested phenotype of
Cdc2-deficient Saccharomyces cerevisiae yeast mutants (5) , and ablation of Cdk2 activity prevents the onset of DNA
replication in mammalian cells(10, 11) . Thus, growth
stimulatory pathways initiated by extracellular signals must ultimately
engage and activate one or more of the G
-specific Cdk
proteins.
Cdk activation is positively regulated by periodic
association with cyclin subunits(2, 3) . Cdk4
complexes with the D-type cyclins, while Cdk2 primarily associates with
cyclin E and cyclin A. Ectopic overexpression of either cyclin D or cyclin E accelerates progression through G
and
reduces the proliferative requirement for serum-derived growth
factors(12, 13, 14) . Conversely, abolition
of cyclin D
or cyclin E activity through the use of
neutralizing antibodies or antisense oligonucleotides effectively
blocks entry into S phase(15, 16) . Ablation of cyclin
A function also prevents DNA replication (17, 18) and
disrupts the checkpoint control pathway that couples mitotic initiation
to the completion of DNA synthesis(19) . However, it appears
that the cyclin A-Cdk2 complex may function at a point distal to
restriction point traverse. Although the mitogen-dependent expression
of cyclin D genes is well defined in mammalian
cells(20, 21, 22) , growth factor regulation
of cyclin E and cyclin A is incompletely understood.
Cyclin
interaction with its catalytic partner is necessary but not sufficient
for kinase activation. Nonfunctional cyclin-Cdk complexes have been
shown to accumulate in serum-stimulated senescent fibroblasts (23) as well as cells that have been growth inhibited by
exposure to TGF-(24) ,
radiation(25) , or
agents which elevate intracellular levels of cAMP(26) .
Activation of assembled cyclin/Cdk holoenzymes is negatively regulated
by direct interaction with Cdk inhibitory proteins, termed
CKIs(27) . Several of these inhibitors function as
intracellular effectors of antiproliferative environmental signals. In S. cerevisiae, cell cycle arrest in response to mating
phermone
is mediated by FAR1, a protein which inactivates the
yeast Cdc2 kinase(28) . In mammalian cells, DNA damaging agents
such as
radiation induce the expression of p53, which
transactivates the promoter of the Cdk inhibitor,
p21
(29) . Furthermore,
TGF-
-dependent inhibition of Cdk2 activation and S phase entry is
mediated by the inhibitor, p27
(30) .
Recently it has been shown that p27 expression is down-regulated after
interleukin 2 stimulation of T lymphocytes(31) , suggesting
that Kip1 may serve as a common target of both positive and negative
growth regulatory pathways.
Kip1 activity may also be regulated
through association with cellular proteins such as Cdk4.
p27 associates with the cyclin D-Cdk4 complex
in a cell cycle-dependent manner (32) , and treatment of human
keratinocytes with the antiproliferative agent TGF-
leads to a
redistribution of p27
from Cdk4 to Cdk2,
correlating with an inhibition of Cdk2 activity and cell cycle
arrest(33) . Baculovirus-produced cyclin D
-Cdk4
complexes can facilitate activation of Cdk2 in
vitro(34) , suggesting that a sequestering of Kip1 by Cdk4
may also be an important component of the mitogenic response to
environmental proliferative signals. However, this hypothesis has not
been rigorously tested in vivo.
Balb/c 3T3 fibroblasts are
a nontransformed mouse cell line that has been extensively
characterized with regard to the proliferative requirements for
specific serum-derived growth factors(34) . Platelet-derived
growth factor (PDGF) acts early in the cell cycle to stimulate the
G to G
transition and render cells competent to
respond to progression factors contained in platelet-poor plasma (PPP).
Sequential exposure of quiescent fibroblasts to PDGF and PPP is
sufficient to stimulate traverse of the restriction point and initiate
commitment to DNA synthesis. Using the Balb/c 3T3 fibroblast system, we
have examined the molecular mechanisms by which growth regulatory
signals such as PDGF and plasma factors cooperatively activate the Cdk2
kinase during late G
. Our results suggest that both a
PDGF-dependent association of Kip1 with Cdk4 and a plasma-dependent
reduction in Kip1 levels are essential for the activation of cyclin E-
and cyclin A-dependent kinases.
Inhibition experiments were performed by mixing extracts of proliferating cells containing active cyclin A-kinase complexes with inhibitor-containing extracts for 1 h at 37 °C prior to immunoprecipitation of cyclin A. Density-arrested Balb/c 3T3 cells were treated with 20 ng/ml PDGF with 10% calf serum in Dulbecco's modified Eagle's medium for 18 h then harvested for proliferating cell extracts. Unless noted otherwise, extracts were mixed in a 1:1 ratio of protein (100 µg:100 µg).
Figure 1: Induction of cyclin A and cyclin E expression and associated kinase activities. Density-arrested Balb/c 3T3 cells were stimulated with 25 ng/ml PDGF-BB or 10% PPP, or both. Whole cell extracts were prepared after a 16-h stimulation for analysis of cyclin E, and after a 21-h stimulation for cyclin A. Top panel, extracts from quiescent and stimulated cells were resolved on a 10% SDS-polyacrylamide gel, immunoblotted, and probed with polyclonal antibodies to cyclin A or cyclin E. Bottom panel, cyclin E and cyclin A immunoprecipitates from nontreated and stimulated cell extracts were analyzed for in vitro histone H1 kinase activity. Phosphorylated proteins were separated on a 10% SDS-polyacrylamide gel and visualized by autoradiography.
Although exposure to PDGF stimulated limited expression of cyclin A protein in nonproliferating cells, it was not sufficient to induce cyclin A-associated kinase activity (Fig. 1). Similarly, cyclin E-Cdk complexes immunoprecipitated from PDGF- or plasma-treated cells failed to phosphorylate histone H1 above basal levels. Identical results were also observed when Cdk2 was immunoprecipitated from fibroblasts treated with PDGF or plasma alone (data not shown). However, cyclin A- and cyclin E-associated kinase activities were dramatically increased in cells receiving both PDGF and PPP. Induction of kinase activity under these conditions was greater than that predicted for an additive response, indicating that plasma-derived progression factors can act synergistically with PDGF to regulate the activation of cyclins A and E associated Cdk2.
Figure 2: Growth factor-stimulated down-regulation of Cdk inhibitory activity. A, density-arrested Balb/c 3T3 cells were stimulated with 10% serum and 10 ng/ml PDGF-BB. After 18 h, extracts containing active cyclin-Cdk complexes were isolated and mixed with inhibitor-containing extracts of quiescent cells. 100 µg of stimulated cell extract were incubated with 100 µg of quiescent cell lysate for 1 h at 37 °C. Cyclin A, cyclin E, Cdk2, and Cdc2 were immunoprecipitated with polyclonal antibodies, and histone H1 kinase activity was determined. Phosphorylated proteins were separated on a 10% SDS-polyacrylamide gel, and kinase activity was quantitated on a PhosphorImager. 100% activity is the activity without inhibitor-containing extracts added. B, density-arrested Balb/c 3T3 cells were stimulated with 25 ng/ml PDGF-BB in the presence and absence of PPP. At the times (hrs) indicated, cells were harvested and whole cell extracts were prepared. Nontreated extracts and extracts heated 5 min at 100 °C were mixed with proliferating cell lysates for 1 h at 37 °C. Cyclin A was immunoprecipitated with a polyclonal antibody, and histone H1 kinase activity was determined. The (+) is without inhibitory extracts added. C, cyclin A-dependent kinase activity shown in B was quantitated using a PhosphorImager, and the percentage of inhibition was graphed. Data points represent the average of three separate experiments. 100% activity is the inhibition produced by quiescent extracts.
Although exposure of quiescent fibroblasts to plasma alone had no effect on inhibitor levels, stimulation of density-arrested Balb/c 3T3 cells with either PDGF or a combination of PDGF and PPP resulted in a nearly identical reduction of free cyclin A/Cdk inhibitory activity that was biphasic in nature (Fig. 2B). Activity of the Cdk inhibitor(s) was rapidly and dramatically reduced by approximately 75% between 2 and 6 h after treatment with PDGF or PDGF/PPP (Fig. 2C). After this time, free inhibitor activity continued to decline at a more gradual rate until it was completely abolished by 12-15 h poststimulation, a point coincident with S phase entry and normal cyclin A activation in those cells exposed to a full complement of growth factors. Down-regulation of free inhibitory activity persisted for at least 24 h after initial mitogen stimulation.
The majority of
Cdk inhibitory activity could be restored to growth factor-treated cell
lysates when samples were heat-treated prior to incubation with
proliferating cell extracts (Fig. 2B). These results
suggest that a Cdk inhibitor present in both quiescent and stimulated
Balb/c 3T3 fibroblasts was reversibly masked by interaction with a
heat-labile factor after exposure to PDGF: p27 had
previously been shown by others to be heat-stable in other
cells(30) . However, boiled extracts of stimulated cells were
less effective in inhibiting the cyclin A-Cdk complex than identically
treated extracts of quiescent cells. Therefore, a decrease in the
abundance of inhibitory factors may also contribute to the apparent
down-regulation of inhibitory activity after growth factor treatment.
Inhibition from boiled extracts decreased slowly starting 6-9 h
after stimulation (Fig. 2C). However, in boiled
extracts from cells treated with PDGF alone, decline in activity was
transient, and the ability to inhibit cyclin A-Cdk complexes returned
to basal levels at later time points. In contrast, total cellular
inhibitory activity continued to decline over the time course of the
experiment when cells were exposed to both PDGF and PPP.
In order to
identify the factor responsible for cyclin A/Cdk inactivation, boiled
lysates from quiescent and stimulated Balb/c 3T3 cells were precleared
with antibodies to p27 prior to use in mixing
experiments. Immunodepletion of Kip1 from extracts of both
nonstimulated cells and cells treated with PDGF and PPP for 24 h
eliminated essentially all inhibitory activity toward the cyclin A-Cdk
complex (Fig. 3A). Immunoprecipitation of
[S
]methionine-labeled cells and Western blot
analysis using the anti-p27 antibody indicated that this antibody did
not recognize p21
or p57
(data not
shown). These data suggest that Kip1 is the primary negative regulator
of cyclin A-dependent kinase activity in Balb/c 3T3 fibroblasts.
Figure 3: Growth factor regulation of Kip1 expression. A, Kip1 protein was immunodepleted from boiled extracts of quiescent and stimulated Balb/c 3T3 fibroblasts (cells were stimulated 21 h in 25 ng/ml PDGF-BB and 10% PPP). Depleted (+) and nondepleted(-) extracts were then mixed with lysates of proliferating cells, and cyclin A-associated histone H1 kinase activity was determined after immunoprecipitation. Control extracts of proliferating cells (lane 1) were mixed with lysis buffer. B, density-arrested cells were stimulated with 25 ng/ml PDGF-BB in the presence and absence of 10% PPP. At the time indicated, extracts were isolated, immunoblotted, and probed with a polyclonal antibody to Kip1. C, Kip1 protein was detected as in B, and levels in cells stimulated 24 h with PDGF or PDGF + PPP were quantitated by laser densitometry. Data points represent the average of three independent experiments.
Immunoblotting of whole cell extracts demonstrated that Kip1 expression was influenced by both PDGF and plasma factors (Fig. 3B). The p27 protein was relatively abundant in quiescent Balb/c 3T3 cells but was moderately reduced in response to treatment with PDGF. The PDGF-mediated reduction in Kip levels reached a nadir 12-18 h after stimulation; however, Kip1 expression increased by 24 h, correlating temporally with a return of total cellular inhibitory activity to the maximal basal level observed in nonstimulated cells (Fig. 2B). In contrast, fibroblasts stimulated with PDGF in the presence of plasma displayed a more dramatic and prolonged reduction in Kip1 expression. By 24 h after stimulation, cells treated with PDGF and PPP contained less than 50% of the p27 expressed in cells receiving PDGF alone (Fig. 3C). Kinetics of the plasma-dependent decline in Kip1 levels at later time points closely paralleled the reduction of inhibitory activity detected in boiled cell lysates. These data show that p27 expression is differentially modulated by PDGF and PPP, and indicate that plasma-derived growth factors are required for a full down-regulation of Kip1 protein.
Although Kip1 levels were dramatically reduced after stimulation with PDGF and PPP, under no condition was Kip1 expression completely abolished. The data presented in Fig. 2indicate that the low level of Kip1 protein present in cells exposed to PDGF and PPP is sufficient to inhibit the majority of cyclin A-dependent kinase activity when released from a masking factor by heat treatment. These results imply that the PDGF-mediated association of p27 with a heat labile silencing factor is likely to be essential for the removal of Kip1 inhibitory activity.
Figure 4:
Effect of cycloheximide on regulation of
Kip1. A, 10 µg/ml cycloheximide (CHX) or 100
µM 5,6-dichlorobenzimidazole (DRB) were added to
density-arrested Balb/c 3T3 cells stimulated with 25 ng/ml PDGF-BB in
the presence and absence of 10% PPP. After 12 h, whole cell extracts
were isolated and mixed with proliferating cell lysates. Cyclin A was
immunoprecipitated from mixed extracts, and histone H1 kinase activity
was determined. Control extracts of proliferating cells were mixed with
lysis buffer (+, lane 1) or nontreated quiescent cell
extract (Q, lane 2). B, density-arrested
cells were treated with PDGF and PPP as above. At the indicated times
after stimulation, cycloheximide was added to culture medium. Cells
represented in lane 8 received no cycloheximide. 12 h after
initial exposure to mitogens, extracts were prepared and mixed with
proliferating cell lysates. Control extracts of proliferating cells
were mixed with lysis buffer (lane 1) or nontreated quiescent
cell extract (Q, lane 2). Cyclin A was
immunoprecipitated from mixed extracts and histone H1 kinase activity
was determined. C, cycloheximide was added to quiescent Balb/c
3T3 cells stimulated with PDGF in the presence or absence of PPP as
described in A. After 12 h, extracts were isolated,
immunoblotted, and probed with polyclonal antibodies Kip1 and cyclin
D.
Identical effects on inhibitory activity were observed in cells that received cycloheximide simultaneously with exposure to PDGF/PPP and those in which cycloheximide was added 2 h after mitogenic stimulation (Fig. 4B). However, down-regulation of free inhibitory activity was markedly less sensitive to protein synthesis inhibition by 4 h after growth factor treatment. Moreover, addition of cycloheximide 4 h prior to harvest only weakly influenced the amount of inhibitory activity. These results, taken together with the data presented in Fig. 4A, indicate that the elimination of free Cdk inhibitory activity is absolutely dependent on protein synthesis 2-4 h after exposure to PDGF.
To determine whether protein
synthesis inhibition affected the growth factor-mediated decrease in
p27 expression, lysates from quiescent Balb/c 3T3 cells stimulated in
the presence and absence of cycloheximide were immunoblotted and probed
with antibodies to Kip1 (Fig. 4C). After a 12 h
stimulation, no detectable difference in Kip1 levels was observed in
cells exposed to cycloheximide and PDGF or PDGF/PPP compared with cells
treated with growth factors alone. In contrast, the addition of
cycloheximide completely abolished PDGF-induced expression of cyclin
D in the same cells. Thus, the effect of cycloheximide on
mitogen-dependent reduction in Cdk inhibitory activity is not mediated
at the level of Kip1 expression. These results demonstrate that the
decline in p27 levels achieved after PDGF stimulation is not sufficient
to effect a decrease in Cdk inhibition.
Figure 5: Inhibitor activity is sequestered by Cdk4 in quiescent Balb/c 3T3 cells. A, various amounts of boiled or nonboiled (no pretreat) quiescent cell extracts were incubated with 100 µg of proliferating cell extract (stimulated 21 h with 25 ng/ml PDGF-BB and 10% PPP). Cyclin A was immunoprecipitated from mixed extracts, and histone H1 kinase activity was determined. Control extracts of proliferating cells (-, lane 1) were incubated with lysis buffer. B, cyclin A-dependent kinase activity measured in A was quantitated using a PhosphorImager and graphed as a percentage of maximum activity. Data points represent the average of three separate experiments. C, normal rabbit serum (NRS) or antibodies to cyclin E, cyclin A, or Cdk4 immobilized on protein A-agarose beads were used in immunoprecipitation of quiescent cell extracts. The beads and the supernatant were separated, boiled, and assayed for inhibition of cyclin A-associated kinase activity.
Since many Cdk inhibitors were originally isolated by virtue of their ability to bind cyclin-Cdk complexes, potential candidate molecules which might sequester p27 in quiescent fibroblasts include the cyclin and Cdk proteins themselves. To test this possibility, extracts of nonstimulated Balb/c 3T3 cells were incubated with either normal rabbit serum or antibodies to cyclin E, cyclin A, or Cdk4 (Fig. 5C). The antibodies were then immobilized on protein A-agarose beads and removed from the lysate by centrifugation. Both the boiled supernatant and the boiled eluate of the immunoprecipitated pellet were then assayed for inhibition toward a cyclin A-Cdk complex. Of the antibodies used, the Cdk4 immunoprecipitate was found to contain the highest level of Cdk inhibitory activity, while a smaller portion of inhibitor was associated with cyclin E. The amount of inhibitory activity released from the Cdk4 complex after heat treatment was comparable to the activity remaining in the supernatant after immunodepletion of the Cdk4 kinase. These results suggest that the majority of sequestered inhibitor in density-arrested Balb/c 3T3 fibroblasts is associated with Cdk4.
In quiescent Balb/c 3T3 fibroblasts, approximately 50% of the
total cellular Kip1 protein is sequestered, while the remainder exists
in a free active state. However, within 12 h after stimulation with
PDGF, all remaining p27 is sequestered. Previously we have demonstrated
that expression of the growth regulatory Cdk4 subunit, cyclin
D, is induced by PDGF during this time period(21) .
As cyclin D
has been reported to associate with the Kip1
inhibitor both in vitro(8) and in
vivo(34) , we examined the role of D
during
PDGF-dependent down-regulation of free inhibitory activity. While Cdk4
levels did not fluctuate during cell cycle progression, cyclin D
was undetectable in quiescent cells and increased dramatically
upon growth factor stimulation (Fig. 6A). D
expression was elevated within 3 h after exposure to PDGF/PPP,
coincident with the onset of a decline in free Cdk inhibitory activity.
Further increases in cyclin D
levels were inversely
proportional to the reduction of Cdk inhibition until after 12 h when
D
levels decreased. Immunoprecipitation of D
after a 12-h exposure to PDGF coprecipitated both the Cdk4 kinase
and p27 (Fig. 6B). These results suggest that the
cyclin D
-Cdk4 complex may regulate the availability of
functional Kip1 protein in growth-stimulated fibroblasts.
Figure 6:
Association of Kip1 and Cdk4 with cyclin
D. A, density-arrested Balb/c 3T3 cells were
stimulated with 25 ng/ml PDGF-BB and 10% PPP. At the times indicated,
whole cell extracts were isolated, immunoblotted, and probed with
polyclonal antibodies to Cdk4 and cyclin D
. B,
quiescent Balb/c 3T3 cells were stimulated with PDGF-BB (25 ng/ml) in
the presence and absence of 10% PPP. 12 h after stimulation, extracts
were prepared and cyclin D
was immunoprecipitated with a
monoclonal antibody covalently linked to agarose. Immunoprecipitates
were resolved on a 10% SDS-polyacrylamide gel, immunoblotted, and
probed sequentially with polyclonal antibodies to cyclin D
,
Cdk4, and Kip1.
To examine
the effect of a cyclin D/Cdk4/Kip1 interaction on free inhibitory
activity, cyclin D and Cdk4 were immunoprecipitated from
extracts of cells stimulated with PDGF or PDGF/PPP for various times.
Both the supernatant and the pellet were then boiled and assayed for
inhibition of cyclin A-dependent kinase activity. As expected, heat
treatment of either the Cdk4 or cyclin D
immunoprecipitates
released an activity that efficiently inactivated the cyclin A-Cdk
complex (Fig. 7A). Cdk inhibitory activity was complexed with
the Cdk4 kinase for at least 21 h after exposure to PDGF. However, a
marked reduction in the amount of inhibitor bound to Cdk4 was observed
in cells stimulated in the presence of plasma, particularly at the
later time points of 18 and 21 h. The identical phenomenon was observed
when cyclin D
was immunoprecipitated from cells treated
with PDGF and PPP.
Figure 7:
Association of Cdk inhibitory activity
with Cdk4 and D in mitogen-stimulated cells. A,
density-arrested Balb/c 3T3 cells were stimulated with PDGF (25 ng/ml)
in the presence and absence of 10% PPP. At the times indicated, whole
cell extracts were isolated, and cyclin D
and Cdk4 were
immunoprecipitated. Antibodies were immobilized on protein A-agarose
beads and removed from the lysate by centrifugation. Pelleted beads
were washed extensively and heated to 100 °C. Eluate of the boiled
pellet was assayed for inhibition of cyclin A-dependent kinase
activity. B, supernatants of cyclin D
and Cdk4
immunoprecipitaions described in A were boiled and assayed for
inhibition of cyclin A/kinase activity. Immunoprecipitations of cyclin
D
and Cdk4 were performed on extracts of quiescent cells
and cells that were stimulated for 21 h. C, kinase activity
measured in A and B at 0 and 21-h time points was
quantitated using a PhosphorImager. Data are expressed as a percentage
of the decrease from control activity immunoprecipitated from
proliferating cell extracts mixed with lysis
buffer.
We next measured the inhibitory activity
remaining in the supernatant after immunodepletion of Cdk4 or cyclin
D. Although a considerable amount of inhibitory activity
associated with Cdk4 in quiescent fibroblasts, sufficient inhibitory
activity remained in the lysate after the removal of either Cdk4 or
D
to maximally inhibit the cyclin A/Cdk enzyme (Fig. 5C and 7B). In contrast, Cdk inhibition
was decreased when cyclin D
or Cdk4 was depleted from
extracts of PDGF-treated cells. However, the most striking reduction of
inhibitory activity was observed after D
or Cdk4 was
cleared from lysates of fibroblasts that were stimulated with PDGF in
the presence of plasma (Fig. 7C). These data
demonstrate that, while Cdk4 interacts with p27 in both quiescent and
stimulated fibroblasts, the cyclin D
-Cdk4 complex has a
greater effect on Kip1 availability in PPP-treated cells due to the
lower abundance of the inhibitor under this condition.
Figure 8:
Cyclin E associated with p27 in quiescent and PDGF treated cells. Density-arrested cells
were treated for 15 h with PDGF (25 ng/ml) with and without 10% PPP.
Equal amounts of protein (10 µg) from extracts of quiescent cells,
cells stimulated with PDGF, and cells treated PDGF with plasma were
analyzed for the amount of cyclin E, and 20 µg of extract were used
to determine cyclin E-Cdk2 histone H1-associated activity as indicated.
The total p27
level was also determine by
direct Western analysis of the extracts (10 µg) along with the
p27
protein found to be associated with cyclin
E by immunoprecipitation of cyclin E (in 40 µg of protein) followed
by Western analysis using anti p27
antibody.
The bottom panel shows the amount of cyclin A-associated
histone kinase inhibitory activity that was associated with the
immunoprecipitated cyclin E (in 40 µg of protein) as determined
after boiling the cyclin E
immunoprecipitate.
Figure 9:
Agents that elevate cAMP inhibit the
induction of cyclin D. Cultures of Balb/c 3T3 cells were
treated with PDGF (25 ng/ml) with or without 10% PPP in the presence or
absence of cholera toxin (CT, 0.5 µg/ml) and
3-isobutyl-1-methylxanthine (IBMX, 10 µM). After
12 h of treatment cells were harvested, extracts were prepared, and
Western analyses were performed with antibodies to cyclin D
(panel A). Panel B shows the amount of
inhibitor activity present in the extracts prepared for panel
A.
Progression through the Balb/c 3T3 cell cycle is regulated by
the sequential and synergistic action of PDGF and plasma-derived
progression factors(34) . While the downstream targets of these
mitogens and their cognate receptors are incompletely defined, it is
clear that the growth regulatory pathways activated by these factors
must ultimately impinge upon the cyclin-dependent kinases and their
cyclin subunits. Previously we have demonstrated that PDGF and other
competence agents which govern the G/G
transition directly engage the cell cycle machinery via
modulation of cyclin D
expression(21) . Here it is
shown that PDGF and plasma factors cooperatively induce the cyclin E-
and cyclin A- dependent kinase activities required for traverse of late
G
and the initiation of DNA replication. Activation of the
PDGF receptor resulted in a limited induction of cyclin E and cyclin A
expression that was not sufficient to overcome the threshold of
inhibition by p27
and allow kinase activation. However,
addition of plasma to PDGF-treated cells stimulated maximal cyclin
expression and an overall reduction of Kip1 levels, thereby promoting
the activation of cyclin associated Cdks. Thus, distinct proliferative
signals from both PDGF and PPP converge upon common targets which
regulate cell cycle progression from G
into S phase in 3T3
fibroblasts. These results provide a molecular basis of how competence
and progression factors might synergistically stimulate cell growth
through unique modulation of the activities of specific Cdk kinases
during the traverse of G
.
The amount of Kip1 available
to bind and inactivate cyclin E- and cyclin A-associated kinases was
regulated in a mitogen-dependent fashion by at least two distinct
mechanisms: 1) active Kip1 protein was sequestered by Cdk4 which
repressed inhibition toward cyclin A-Cdk complexes in lysate mixing
experiments, and 2) total protein levels of Kip1 were decreased.
Reduction of Kip1 expression occurred in two phases that were
differentially regulated by PDGF and PPP. Exposure of quiescent
fibroblasts to PDGF stimulated a moderate decline in p27 levels that
began within 6 h of mitogen stimulation. Although in several cell types
Kip1 expression is elevated in response to antiproliferative signals
such as TGF-(33) , this early component of Kip1
elimination was not affected by inhibitors of Balb/c 3T3 cell growth
such as cAMP and cycloheximide. However, the PDGF-mediated removal of
free Cdk inhibitory activity was prevented under conditions of protein
synthesis inhibition. Therefore, the down-regulation of Kip1 levels
achieved after treatment with PDGF alone was not sufficient to ablate
inhibition of the cyclin A/Cdk enzyme as determined in the in vitro assays. In contrast, stimulation of PDGF-treated cells with PPP
resulted in a more pronounced decline in p27 expression, particularly
at later time points when cyclin A-associated kinase activity was
maximal. Thus, the greatest decrease in p27 was observed under
conditions that stimulated DNA synthesis. This plasma-dependent
reduction of Kip1 levels, together with the PDGF-mediated inactivation
of Kip1 by Cdk4, critically limited the interaction of Cdk inhibitors
with cyclin E and cyclin A kinase partners.
Dissociation of Kip1
from labile proteins after heat treatment revealed that enough
inhibitor was present to reduce cyclin A-dependent kinase activity by
70-80%, even after maximal down-regulation of Kip1 expression in
plasma-treated cells. Therefore, sequestering of free Kip1 protein
after growth factor stimulation is likely to be essential for the
activation of cyclin E- and cyclin A-associated kinases. Previously it
has been shown that cyclin D-Cdk4 complexes compete with cyclin E-Cdk2
and cyclin A-Cdk2 for binding of the Kip1 inhibitor in
vitro(30) . These data suggest that growth factor-mediated
assembly of cyclin D/Cdk4 holoenzymes and consequent association with
p27 may facilitate activation of Cdk2 later in the cell cycle.
Consistent with this hypothesis, treatment of epithelial cells with
TGF- elevates the synthesis of p15 which displaces Kip1 from Cdk4.
p27
is then available to bind and inhibit
Cdk2(33) . In Balb/c 3T3 fibroblasts stimulated with PDGF
either in the presence or absence of plasma, a substantial proportion
of Kip1 was associated with Cdk4. As a consequence of this Kip1/Cdk4
interaction, the cellular pool of inhibitory activity was depleted by
greater than 50%. Our results suggest that Cdk4 is an integral
component of a mitogen-stimulated feed-forward mechanism which promotes
activation of Cdk2 in Balb/c 3T3 cells.
The affinity of Cdk4 for
Kip1 in vitro is increased by association with a cyclin
subunit(36) . Cdk4 assembles combinatorially with
three-dimensional-type cyclins which are differentially induced in
various cell types. Transcripts for all three of the D cyclins are
expressed during the G phase of the Balb/c 3T3 cell cycle;
however, only cyclin D
is up-regulated in response to PDGF (21) . The kinetics of cyclin D
increase temporally
correlated with a reduction in free inhibitor levels after PDGF
stimulation. Furthermore, D
expression was first detected
during a window of time when down-regulation of inhibitory activity is
absolutely dependent on new protein synthesis. As cyclin D
levels increased, removal of Cdk inhibitory activity became less
sensitive to protein synthesis inhibition. Immunoprecipitation of
cyclin D
during peak expression coprecipitated Kip1
protein, and boiling of the immunoprecipitate released a considerable
amount of Cdk inhibitory activity. Comparison of D
and Cdk4
immunoprecipitates revealed a nearly identical pattern of association
with the inhibitor, suggesting that Cdk4 modulation of Kip1
availability in growth-stimulated cells was primarily effected by
complexes containing cyclin D
.
However, down-regulation
of Cdk inhibition may not be strictly dependent on cyclin
D. Treatment of Balb/c 3T3 cells with cAMP-inducing agents
inhibited D
expression, but only weakly antagonized the
removal of free Cdk inhibitory activity after PDGF stimulation.
Presently, it is not known whether Cdk4 expressed under these
conditions sequesters Kip1 in association with another cyclin partner,
or whether the small amount of D
induced in the presence of
cAMP is sufficient to modulate Kip1 availability. However, Cdk4 was
found to bind a large amount of inhibitory activity in quiescent cells
despite the absence of cyclin D
protein. Transcripts for
both cyclin D
and cyclin D
are relatively
abundant in density-arrested Balb/c 3T3 fibroblasts(21) , and
cyclin D
-Cdk4 complexes compete more effectively for Kip1
binding in vitro than do cyclin D
-Cdk4
complexes(36) . Thus, the Cdk4/p27 interaction may be directed
by various cyclin partners during different stages of the cell cycle.
One consequence of Kip1 association with Cdk4 in quiescent cells may be
to maintain Cdk4 in an inactive state until normal cell cycle
progression is initiated in response to growth factor stimulation.