The EMBO Journal Vol.18 No.6 pp.1571–1583, 1999
The p21Cip1and p27Kip1CDK ‘inhibitors’ are essential
activators of cyclin D-dependent kinases in murine
Mangeng Cheng1, Paul Olivier2,3,
J.Alan Diehl1,3, Matthew Fero2,3,
Martine F.Roussel1, James M.Roberts2,3and
1Department of Tumor Cell Biology, St Jude Children’s Research
Hospital, 332 N. Lauderdale, Memphis, TN 38105,2Division of Basic
Sciences, Fred Hutchinson Cancer Center, Seattle, WA 98104 and
3Howard Hughes Medical Institute, USA
The widely prevailing view that the cyclin-dependent
kinase inhibitors (CKIs) are solely negative regulators
of cyclin-dependent kinases (CDKs) is challenged here
by observations that normal up-regulation of cyclin D–
CDK4 in mitogen-stimulated fibroblasts depends
redundantly upon p21Cip1and p27Kip1. Primary mouse
embryonic fibroblasts that lack genes encoding both
p21 and p27 fail to assemble detectable amounts of
cyclin D–CDK complexes, express cyclin D proteins at
much reduced levels, and are unable to efficiently
direct cyclin D proteins to the cell nucleus. Restoration
of CKI function reverses all three defects and thereby
restores cyclin D activity to normal physiological levels.
In the absence of both CKIs, the severe reduction in
cyclin D-dependent kinase activity was well tolerated
and had no overt effects on the cell cycle.
Keywords: CDK4/cell cycle/D-type cyclins/p21Cip1/
Regulation of mammalian cell proliferation by extracellu-
lar mitogens is governed through receptor-mediated sig-
naling circuits which ultimately converge on the cell cycle
machinery driven by cyclin-dependent kinases (CDKs)
1995). One important example is receptor-activated Ras
signaling, which governs the accumulation of cyclin D1–
mentary pathways: gene transcription, protein association
and protein stabilization. First, Ras signaling promotes
transcription of the cyclin D1 gene via a kinase cascade
that depends upon the sequential activities of Ras, Raf-1,
mitogen-activated protein kinase kinase (MEK1) and
mitogen-activated protein kinases (MAPKs), also referred
to as extracellular signal-regulated protein kinases (ERKs)
(Albanese et al., 1995; Lavoie et al., 1996; Winston et al.,
1996; Aktas et al., 1997; Kerkhoff and Rapp, 1997; Weber
et al., 1997). Signaling through this same pathway is
also sufficient to promote assembly of cyclin D1–CDK4
complexes (Cheng et al., 1998), although the physiological
target of ERK phosphorylation that mediates this process
© European Molecular Biology Organization
has not been defined. Finally, proteasomal degradation of
cyclin D1 is triggered by its phosphorylation on a single
threonine residue (Thr286) by glycogen synthase kinase-
3β (Diehl et al., 1997, 1998), a process antagonized
by signaling through a separate Ras-dependent pathway
kinase B (also called Akt) (Boudewijn et al., 1995; Cross
et al., 1995; Franke et al., 1995, 1997; Klinghoffer et al.,
1996; Dudek et al., 1997; Vanhaesebroeck et al., 1997).
In the continued presence of mitogenic signals, cyclin D1–
CDK4 complexes assemble and accumulate throughout G1
phase, enter the nucleus and undergo phosphorylation by
CDK-activating kinase (CAK) to yield active holoen-
is to initiate phosphorylation of the retinoblastoma protein
(Rb), thereby helping to cancel its activity as a transcrip-
tional repressor of a bank of genes, including cyclins E
and A, whose activities are required for S phase entry
(Weinberg, 1995; Sherr, 1996).
A separate, non-catalytic action of cyclin D-dependent
kinases is the sequestration of CKIs, including p27Kip1
and p21Cip1(Sherr and Roberts, 1995). The Cip/Kip
proteins interact with a variety of cyclin–CDK complexes
through a conserved N-terminal domain that contains both
cyclin and CDK binding sites (Toyoshima and Hunter,
1994; Chen et al., 1995, 1996; Luo et al., 1995; Nakanishi
et al., 1995; Lin et al., 1996; Russo et al., 1996). Cyclin
D-dependent CDKs isolated from mammalian cells appear
to be less susceptible to Cip/Kip-mediated inhibition than
are other classes of cyclin–CDKs (Soos et al., 1996; Blain
et al., 1997; LaBaer et al., 1997), and sequestration of
p21Cip1and p27Kip1into higher order complexes with
cyclin D-dependent kinases during G1phase helps to
relieve cyclin E–CDK2 from their constraint, thereby
facilitating its activation later in G1phase. This ability
to ‘titrate’ CKIs therefore sets a dependency of cyclin
E-dependent kinase on the mitogen-stimulated assembly of
cyclin D-dependent kinases. Cyclin E–CDK2 collaborates
with cyclin D-dependent kinases to phosphorylate Rb
(Hatakeyama et al., 1994; Mittnacht et al., 1994; Lee
et al., 1996; Kelly et al., 1998; Lundberg and Weinberg,
1998), phosphorylates p27Kip1to trigger its degradation
(Sheaff et al., 1997; Vlach et al., 1997), and may target
other proteins whose modifications trigger origin firing
and DNA replication per se (Stillman, 1996; Krude
et al., 1997).
Although it is generally assumed that CKIs act solely
to retard G1progression, the fact that they can be found
in complexes with active cyclin–CDKs (Zhang et al.,
1994; Soos et al., 1996; Blain et al., 1997; LaBaer et al.,
1997) raises the possibility that they may also act as
positive regulators. Intriguingly, LaBaer et al. (1997)
demonstrated that p21Cip1could promote the assembly of
active cyclin D1–CDK4 complexes and, in addition, could
M.Cheng et al.
provide a localization signal for their nuclear import.
However, the fact that p21 nullizygous mice undergo
normal development and do not seem to have a significant
(Brugarolas et al., 1995; Deng et al., 1995) leaves open
the question of whether the CKIs are normal physiological
regulators of cyclin D–CDK assembly. In this study, we
have used primary mouse embryo fibroblast (MEF) strains
deficient in p21, p27 or both to study their roles in
governing the activities of cyclin D–CDK holoenzymes.
Impaired assembly of cyclin D–CDK4 complexes in
MEFs lacking p21 and p27
Cell lysates from asynchronously proliferating MEFs
derived from wild-type mice, p21- and p27-null mice and
from animals lacking both genes were precipitated with
antibodies to cyclin D1 or CDK4. Precipitated proteins
were resolved on denaturing polyacrylamide gels, trans-
ferred to nitrocellulose membranes and blotted with the
cognate antibodies to quantitate cyclin D1 and CDK4
levels, respectively, or with the reciprocal antibodies to
score for the presence of cyclin D1–CDK4 complexes
(Figure 1A). In early passage (p5) wild-type MEFs, ~40%
of the total CDK4 (lane 1, K4 blot) co-precipitated with
antibodies to D1 (lane 2, K4 blot). A smaller percentage
of the total D1 pool (lane 2, D1 blot) co-precipitated with
CDK4 (lane 1, D1 blot). However, antibodies to full-length
recombinant CDK4 used in this experiment preferentially
detect free versus cyclin D1-bound catalytic subunits, so
the amount of D1 detected in CDK4 immunoprecipitates
underestimates the extent of complex formation. Generally
equivalent levels of CDK4 were expressed in MEF strains
lacking one or both CKIs (Figure 1A, lanes 3, 5 and 7). In
contrast, the overall levels of cyclin D1 were significantly
lowered in cells lacking either p21 or p27 (D1 blot, lanes
6 and 8 versus lane 2) and were decreased at least 10-
fold in lysates of cells lacking both CKIs (lane 4). Cyclin
D1–CDK4 complexes were recovered at similarly reduced
levels from lysates of p21-null and p27-null MEFs (lanes
5–8), but at this level of resolution, no cyclin D1–CDK4
complexes were detected in immune precipitates from
cells lacking both CKIs (lanes 3 and 4).
Reduced cyclin D1–CDK4 complex formation in
double-null MEFs may have simply reflected the lower
levels of cyclin D1 expressed in these cells. However,
several lines of evidence indicate that this is not the
explanation. First, when cell lysates were normalized so
that each contained comparable amounts of cyclin D1
protein, the levels of CDK4 that co-precipitated with
cyclin D1 were again found to be decreased in p21-null
or p27-null cells (Figure 1B, lanes 2 and 3) and were very
much reduced in p21/p27 double-null MEFs, whether the
cells were in early (p7) or late (p15) passage (Figure 1B,
lanes 4 and 6). We estimated that p7 double-null cells
contained ?10% of the D1–CDK4 complexes detected
in age-matched wild-type MEFs. Secondly and more
importantly, infection of p21/p27 double-null MEFs for
48 h with a retrovirus encoding Flag epitope-tagged cyclin
D1 restored high levels of D1 expression but not D1–
CDK4 complex formation (Figure 1C). This demonstrated
directly that the lower levels of cyclin D1 expressed in
Fig. 1. Impaired assembly of cyclin D–CDK4 complexes in MEFs
lacking p21 and p27. (A) Cell lysates (500 µg total protein per lane)
from MEFs of the indicated genotypes were immunoprecipitated (IP)
with antibodies to CDK4 (K4) or cyclin D1 and the separated proteins
were blotted with the cognate or reciprocal antibodies. (B) Cell lysates
were normalized for cyclin D1 abundance, and D1 immune
precipitates were blotted with antibodies to cyclin D1 or CDK4. Two
electrophoretic forms of cyclin D1 detected in this experiment can be
routinely observed when separation conditions are sufficiently stringent
(Matsushime et al., 1991); both are phosphoproteins and the nature of
the differences between them remains unclear (Diehl et al., 1998).
(C) Wild-type or p21/p27 double-null MEFs were infected with
retrovirus encoding Flag-tagged cyclin D1. Cell lysates prepared 48 h
post-infection were precipitated with a control monoclonal antibody
(C) or with antibodies to the Flag epitope (M2), and the separated
proteins were blotted with antibodies to cyclin D1 or CDK4. (D) Cell
lysates from MEFs with the indicated genotypes were precipitated
with antibodies to cyclin D2 and the separated proteins were blotted
with antibodies to cyclin D2 or CDK4. All immunoblots were
visualized using enhanced chemiluminescence.
the p21/p27 double-null cells do not account for the defect
observed in cyclin D1–CDK4 assembly.
MEFs also express cyclin D2 (Figure 1D, lane 1) but
little detectable cyclin D3 (data not shown). Compared
with wild-type MEFs, cyclin D2 levels were reduced by
~30% in cells lacking either p21 (lane 2) or p27 (lane 3)
CDK inhibitors promote cyclin D–CDK assembly
Fig. 2. Cyclin D1 and CDK4-dependent Rb kinase activity in MEFs
lacking p21 and p27. (A) Lysates from MEFs of the indicated
genotypes were precipitated with non-immune rabbit serum (NRS) or
with antibodies to cyclin D1 or CDK4. Resulting complexes were
assayed for kinase activity using GST-Rb as the substrate. (B) Lysates
from cells of the indicated genotype were depleted of p21, p27 or
both, and then precipitated with antibodies to CDK4 or control NRS.
Washed immune complexes were assayed for Rb kinase activity.
and, like D1, were significantly decreased in cells lacking
both CKIs (lane 4). Despite the fact that the D2 signal in
Figure 1D exceeds the D1 signal in Figure 1A and
B, quantitation of the two cyclins by comparison with
recombinant protein standards indicated that the level of
D1 exceeds that of D2 by 2- to 3-fold (data not shown).
Cyclin D2–CDK4 complexes were readily detected in
wild-type MEFs and in those lacking either CKI but
were significantly reduced in cells lacking both inhibitors
(Figure 1D, K4 blot). In agreement with previous data
showing that CDK4 is the predominant partner of D-type
cyclins in rodent fibroblasts (Matsushime et al., 1994),
virtually no cyclin D1–CDK6 or D2–CDK6 complexes
were detected in MEFs.
D-type cyclin binding is essential for activating CDK4
of D-type cyclins with CDK4 was significantly compro-
mised in the p21/p27 double null MEFs, both cyclin D1-
dependent and total CDK4 kinase activity were measured
in these cells (Figure 2A). Cell lysates from proliferating
MEFs were precipitated with either non-immune rabbit
serum (NRS), antibody to cyclin D1 or antibody to CDK4,
and the resulting immune complexes were assayed for
kinase activity using recombinant GST-Rb as the substrate.
Note that CDK4-dependent kinase activity should include
contributions from both D1- and D2-containing holo-
enzymes (Figure 1). Active cyclin D1- and CDK4-depend-
ent Rb kinase activities were detected in precipitates from
wild-type MEFs (Figure 2A, lanes 2 and 6), p21-null
MEFs (lanes 3 and 7) and p27-null MEFs (lanes 4 and
8). In contrast, only background levels of kinase activity
were detected in immune complexes recovered from p21/
p27 double-null MEFs (lanes 5 and 9), consistent with
observations that few cyclin D–CDK4 complexes were
formed (Figure 1).
Cell lysates from wild-type MEFs were subjected to
two rounds of immunodepletion using antisera to p21,
p27 or to both, and CDK4 kinase activity was measured
using glutathione S-transferase (GST)-Rb as the substrate.
Removal of p27 (Figure 2B, lane 3) or p21 (lane 4) from
lysates of wild-type MEFs partially reduced CDK4 kinase
activity, whereas elimination of both p21 and p27 (lane
5) completely depleted the activity. Similarly, removal of
p27 from lysates of p21-null MEFs (lane 7) or vice versa
(lane 9) depleted all CDK4 kinase activity from these
lysates. Therefore, both p21- and p27-associated cyclin
D–CDK complexes retain activity (Zhang et al., 1994;
Soos et al., 1996; Blain et al., 1997; LaBaer et al., 1997),
consistent with results that either p21 or p27 is required for
efficient assembly of active cyclin D–CDK4 complexes.
p21 or p27 promotes assembly of stable
cyclin D1–CDK4 complexes in double-null MEFs
One prediction is that reintroduction of p21 or p27 into
double-null MEFs should increase the assembly of cyclin
D1–CDK4 complexes. We infected these cells either with
a control retrovirus encoding the T-cell co-receptor CD8
or with retroviruses encoding either p21 or p27 (Figure
3A). Lysates prepared from MEFs infected for 48 h were
precipitated with antibodies to cyclin D1 or CDK4, and
assayed for complex formation. Ectopic expression of
either p21 (Figure 3A, lane 3) or p27 (lane 4) but
not CD8 (lane 1) increased cyclin D1–CDK4 complex
formation in p21/p27 double-null MEFs. The N-terminal
portion of p27, which contains the cyclin and CDK binding
sites, is sufficient to promote the stable association of
cyclin D1 and CDK4, whereas the C-terminal half of p27
is inactive in this assembly assay (data not shown). Ectopic
expression of another CDK inhibitor, p16INK4a, which
binds to CDK4 or CDK6 but not to D cyclins (Serrano
et al., 1993), did not promote assembly of cyclin D1–
CDK4 in these cells (data not shown). Ectopic expression
of p21 or p27 not only increased the assembly of cyclin
D–CDK4 complexes but also increased the overall levels
of cyclin D1 in p21/p27 double-null MEFs to levels that
approached those in wild-type MEFs (Figure 3A, lanes 3
and 4 versus lane 2).
Cyclin D1 is a labile protein (Matsushime et al.,
1992), and its rapid proteolytic degradation is triggered
by phosphorylation on Thr286 (Diehl et al., 1997). By
interacting with both cyclin D1 and CDK4, p21 and p27
might slow cyclin D1 turnover, possibly by promoting
nuclear localization of the complexes (see below) and/or
by interfering with cyclin D1 phosphorylation. Double-
null MEFs were infected for 48 h with a retrovirus
encoding p27, and cells were metabolically labeled with
[35S]methionine for 30 min. Medium containing labeled
methionine was removed, and cells were incubated in
complete medium containing a 100-fold excess of un-
labeled methionine. Cell lysates prepared after different
periods of ‘chase’ were precipitated with the monoclonal
antibody to cyclin D1, and the labeled proteins were
resolved on a denaturing gel (Figure 3B). The half-life of
cyclin D1 in p21/p27 double-null MEFs in several such
experiments was calculated to be 15 min (Figure 3B, lanes
M.Cheng et al.
Fig. 3. Reconstitution of cyclin D1–CDK4 complexes in vivo and
restabilization of cyclin. (A) Wild-type MEFs or those lacking both
p21 and p27 were infected for 48 h with control virus (CD8) or with
viruses encoding p21 or p27. Cells were lysed and immunoprecipitated
with antibodies to cyclin D1 or CDK4. Separated immune complexes
were then blotted with the cognate or reciprocal antibodies, and sites
of antibody binding were detected by enhanced chemiluminescence.
(B) MEFs lacking both p21 and p27 were infected with a control
retrovirus encoding CD8 or with a virus encoding p27. Two days post-
infection, cells were pulse-labeled for 30 min with [35S]methionine
and then ‘chased’ in the presence of 100-fold excess of unlabeled
methionine for the indicated times. Lysates normalized for protein
concentration were precipitated with a monoclonal antibody to cyclin
D1, and the labeled proteins were resolved on a denaturing gel, which
was dried and subjected to autoradiography.
2–5), which is shorter than that in wild-type cells (t1/2?
25 min) (Matsushime et al., 1992; Diehl et al., 1997,
1998). In contrast, in cells infected with p27-virus, the
half-life of D1 exceeded 40 min (Figure 3B, lanes 6–
9). Metabolic labeling experiments indicated that the
relatively low level of cyclin D1 in p21/p27 double-null
MEFs also reflects a 3-fold reduced rate of D1 synthesis
versus that in wild-type MEFs (data not shown). Cyclin
D1 synthesis was only modestly increased following acute
infection of the cells with the p27 retrovirus (Figure 3B,
compare lane 6 with lane 2), so the restoration of D1
levels following reintroduction of p21 or p27 (Figure 3)
primarily reflects increased D1 stability.
Association of CDK4 with Cdc37 and INK4 proteins
in p21/p27 double-null cells
Although CDK4 in mouse fibroblasts has a half-life of
~4 h (Matsushime et al., 1992), unassembled CDK4
subunits are unstable, and the levels of monomeric CDK4
are very low (Dai et al., 1996; Stepanova et al., 1996).
Similarly, the vast majority of CDK6 subunits are bound
to other molecules (Mahony et al., 1998). CDK4 requires
association with Hsp90/Cdc37 for stabilization, suggesting
that the latter acts as a chaperone for the proper folding
of kinase subunits (Dai et al., 1996; Stepanova et al.,
1996). High molecular weight complexes containing
Hsp90, Cdc37 and CDK4 or CDK6 are cytoplasmic and
do not contain D-type cyclins, so assembly of cyclin D
with CDK4, the nuclear translocation of these complexes,
and their activation by CAK presumably occur as later
null cells, the lack of complexes between CDK4 and
cyclins D1 and D2 might result in a greater association
of CDK4 subunits with Cdc37. Instead, the level of Cdc37-
bound CDK4 was lower in double-null cells than in wild-
type cells (Figure 4D), suggesting that most CDK4 was
complexed with other molecules or remained monomeric.
Apart from interacting with D-type cyclins, CDK4 and
CDK6 can independently associate with INK4 proteins
(Serrano et al., 1993; Guan et al., 1994; Hannon and
Beach, 1994; Chan et al., 1995; Hirai et al., 1995). CDK–
INK4 complexes are stable, lack Cdc37, cannot assemble
with cyclins, and therefore appear inaccessible for enzym-
atic activation (Parry et al., 1995; Stepanova et al., 1996).
It is therefore presumed that CDK4 can achieve alternative
fates after release from the chaperone complex, either
assembling with D-type cyclins or being inactivated
through INK4 binding. Possibly, p21 and p27 might
facilitate assembly of CDK4 and D-type cyclins by
blocking the ability of INK4 proteins to sequester CDK4
in an inactive pool. We therefore studied the expression
of the four different INK4 family members (p16INK4a,
p15INK4b, p18INK4cand p19INK4d) and compared their
associations with CDK4 in wild-type and p21/p27 double-
by two sequential immunoprecipitations, and the levels of
CDK4 associated with each INK4 family member were
determined by blotting the precipitated proteins with
antibodies to CDK4 (Figure 4A, lanes 1 and 2). Lysates
depleted of individual INK4 proteins were then precipit-
ated and blotted with antibodies to CDK4, in order
to estimate the levels of residual CDK4 that remained
unassociated with INK4 proteins (Figure 4A, lanes 3).
Wild-type MEFs expressed significant amounts of CDK4
in complexes with p16INK4a, p15INK4band p18INK4c(Figure
4A). In cells lacking both p21 and p27, CDK4 associated
with the same three INK4 family members, although in
comparison with wild-type MEFs, less CDK4 was bound
to p18INK4cand more was bound to p15INK4b(Figure 4B).
In both MEF strains, more CDK4 was complexed with
p16INK4athan with other INK4 family members, and no
association with p19INK4dwas detected.
To determine the relative pools of total INK4-bound
and -unbound CDK4, MEF lysates were depleted with
mixtures of antibodies directed to all four INK4 family
members, and the above analysis was repeated (Figure
4C). From several such experiments, we estimated that in
wild-type cells, ~40% of the total CDK4 pool stably
associated with INK4 proteins. As expected, cyclin D1
co-precipitated only with those CDK4 molecules that
were not bound to INK4 proteins (?/? cells, lanes 3).
CDK inhibitors promote cyclin D–CDK assembly
Fig. 4. Association of CDK4 with INK4 proteins and Cdc37. Cell lysates from wild-type (A) or p21/p27 double-null cells (B) were sequentially
depleted by two rounds of precipitation with non-immune rabbit serum (NRS) or with antibodies to the designated INK4 proteins (lanes 1 and 2)
and then precipitated with anti-CDK4 (lanes 3). All recovered proteins were immunoblotted with anti-CDK4. (C) Experiments were performed as
above, except that immunodepletion was carried out with a mixture of antibodies to all four INK4 family members prior to blotting of precipitated
proteins with anti-CDK4 and anti-D1. (D) Cells of the indicated genotype were lysed and equal quantities of protein (100 µg) were resolved on
denaturing gels and immunoblotted directly with anti-CDK4 (lanes 1 and 2) or rabbit anti-Cdc37 (lanes 5 and 6; produced by J.A.D. and C.J.S. to
recombinant mouse protein synthesized in bacteria, unpublished). The arrows indicate the position of authentic CDK4 (34 kDa, left) and Cdc37
(50 kDa, right). The faster-migrating band detected with commercial polyclonal antibodies to mouse CDK4 used in this experiment (Santa Cruz
Biotechnology) is not observed using antisera raised in our laboratory (RYor RZ; Matsushime et al., 1994). To quantitate complex formation, 5-fold
more lysate protein (500 µg/lane) precipitated with anti-Cdc37 was separated on denaturing gels and blotted with anti-CDK4 (lanes 3 and 4).
Importantly, all enzymatically active CDK4 is bound to
D-type cyclins, and this fraction also contained associated
p21 and p27 molecules (Figure 2). In p21/p27 double-
null cells, the INK4-bound CDK4 fraction was increased
to ~50–60%; D1–CDK4 complexes were again not
detected even though a substantial pool of non-INK4-
bound CDK4 remained (–/– cells, lanes 3). Therefore, in
wild-type MEFs, ~40% of CDK4 is associated with cyclin
D1 and 10–15% is associated with D2 (Figure 1), ~40%
is associated with INK4 proteins (Figure 4C), and much
of the remainder is bound to Cdc37 (Figure 4D; see figure
legend for amounts of protein loading per lane). In p21/
p27 double-null cells, little CDK4 binding to D cyclins
was detected (Figure 1), ~60% was bound to INK4
proteins (Figure 4C) and ?10% was complexed to Cdc37.
Therefore, a substantial fraction of CDK4 must either
remain monomeric or is associated with as yet unidentified
molecules. This indicates that p21 and p27 do not simply
compete with INK4 proteins in directing cyclin D–CDK
p21 or p27 can facilitate nuclear accumulation of
Cyclin D1 normally accumulates in the nuclei of cells
during G1phase but relocalizes to the cytoplasm during
S phase (Baldin et al., 1993). Although cyclin D1 has no
obvious nuclear import signal, p21 family members can
direct the nuclear localization of cyclin D1–CDK4 com-
plexes (Diehl and Sherr, 1997; LaBaer et al., 1997),
raising the possibility that cyclin D1 might not be able to
enter the nucleus in MEFs lacking both p21 and p27.
When asynchronously proliferating wild-type MEFs were
Table I. Subcellular localization of cyclin D1 in cells lacking CKIs
Genotype% Nuclear % Nuclear and
61 ? 4
49 ? 4
46 ? 4
38 ? 3
21 ? 3
23 ? 3
26 ? 4
28 ? 3
18 ? 3
28 ? 4
28 ? 4
34 ? 4
Flag-tagged D1 retrovirus
59 ? 5
7 ? 3
23 ? 2
64 ? 5
18 ? 2
29 ? 4
Flag-tagged D1 (T286A) retrovirus
82 ? 5
77 ? 7
14 ? 2
17 ? 5
4 ? 1
6 ? 2
Proliferating wild-type MEFs and those lacking p21, p27 or both were
stained with monoclonal antibody to cyclin D1 and scored by
immunofluorescence for the presence of nuclear and/or cytoplasmic
D1. Cells infected with retroviruses encoding Flag-tagged D1 or the
D1 (T286A) mutant that is stable and remains in the nucleus
throughout interphase were studied similarly. Because ectopic
expression of D1 greatly exceeded that of the endogenous protein, no
background signals were detected at the exposures used. At least 500
cells were counted per experiment and the results show mean ? SD
from three such experiments.
stained with antibody to cyclin D1, 61% of the cells
exhibited strong nuclear fluorescence (Table I). Fluores-
cence-activated cell sorter (FACS) analysis of DNA con-
tent indicated that 52% of the total asynchronously
proliferating population were in G1phase, and in agree-
M.Cheng et al.
Fig. 5. Assembly of ectopically expressed cyclin D1 and D1 (T286A)
with CDK4. Wild-type and double-null MEFs were infected with
retrovirus encoding Flag-tagged cyclin D1 or D1 (T286A) for 48 h.
Lysates were then precipitated with a control monoclonal antibody (C)
or with the monoclonal antibody to the flag epitope (M2), and the
resulting precipitates were resolved on a denaturing gel and transferred
to nitrocellulose. Proteins were visualized by enhanced
chemiluminescence using a monoclonal antibody to D1 or antibodies
ment with others’ results (Baldin et al., 1993), double-
labeling with BrdU for 2 h prior to staining indicated
that ~90% of cells exhibiting bright nuclear cyclin D1
fluorescence were not in S phase (data not shown).
Although a smaller fraction of cells lacking p21, p27 or
both exhibited exclusively nuclear staining, the lower
levels of cyclin D1 expressed were still able to enter the
nucleus (Table I). In agreement with immunoblotting
results (Figure 1), the intensity of cyclin D1 staining in
p21/p27 double-null MEFs was much lower than that of
wild-type MEFs (as judged by the need for a 6-fold
increase in exposure time to obtain an almost comparable
signal). Hence, the CDK inhibitors are not strictly required
for cyclin D1 nuclear import. Moreover, the fact that a
significant fraction of cyclin D1 was detected in the
nucleus of double-null cells (Table I) whereas ?95%
failed to assemble with CDK4 (Figure 1) suggests that
stable association with catalytic subunits is also not
essential for D1 nuclear import.
We next used infection with a retrovirus encoding Flag-
tagged D1 to increase cyclin D levels in double-null cells.
After infection for 36 h, similar amounts of Flag-D1 were
expressed in both wild-type and the double-null MEFs,
as demonstrated by immunoprecipitation with M2 anti-
bodies to the tag followed by immunoblotting with anti-
bodies to D1 (Figure 5, lanes 2 versus 4, D1 blot). These
levels of ectopically expressed cyclin D1 exceeded the
endogenous level of cyclin D1 in double-null MEFs by
?10-fold (data not shown). Most infected wild-type MEFs
(59%) displayed an exclusively nuclear cyclin D1 staining
pattern (Table I). In marked contrast, ?7% of p21/p27
double-null MEFs exhibited exclusively nuclear cyclin D1
staining, and instead, the cells displayed both nuclear and
cytoplasmic staining or exclusively cytoplasmic staining
(Table I). Therefore, under conditions in which cyclin D1
was restored, an absence of p21 and p27 limited D1
nuclear accumulation. Importantly, ectopically expressed
Flag-tagged cyclin D1 was still unable to assemble with
CDK4 in cells lacking both p21 and p27 (Figure 5, lane
4, CDK4 blot; also see Figure 1C).
These experiments left open the possibility that the
ability of p21 and p27 to promote assembly of cyclin D1–
CDK4 complexes was an indirect effect of their directing
cyclin D1 to the nucleus. Accordingly, we took advantage
of a mutant form of cyclin D1 (T286A) that contains an
alanine for threonine-286 substitution, is remarkably stable
(t1/2?3 h) (Diehl et al., 1997), and remains in the nucleus
throughout the cell cycle (Diehl et al., 1998). When
infected with a vector encoding D1 (T286A), both wild-
type MEFs (82%) and p21/p27 double-null MEFs (77%)
displayed an exclusively nuclear staining pattern of the
D1 mutant (Table I), reinforcing the concept that D1 can
enter the nucleus in the absence of both p21 and p27.
Moreover, the abundance of cyclin D1 (T286A) was
similar in both wild-type and double-null MEFs (Figure
5, lanes 3 and 5).Even under these conditions, D1 (T286A)
could not assemble with CDK4 (Figure 5, lane 5, CDK4
blot). This argues that CKIs do not ensure assembly by
simply contributing a nuclear import signal, in agreement
with results obtained with mutants of p21 (LaBaer et al.,
1997) and p27 (not shown) that lack the signal sequences.
These results also confirm that decreased assembly of
cyclin D1–CDK4 complexes is not simply secondary to
the decreased abundance of cyclin D1 in p21/p27 double-
Rb phosphorylation in p21/p27 double-null cells
In normal MEFs, phosphorylation of Rb is triggered by
cyclin D-dependent kinases and is probably completed by
cyclin E–CDK2 (and/or cyclin A–CDK2) as cells enter S
phase. Since active cyclin D-dependent kinase activity
was not detected in the p21/p27 double-null MEFs (Figure
2), we studied the kinetics of Rb phosphorylation in these
cells. MEFs arrested by contact inhibition and serum
starvation were trypsinized and reseeded at lower density
in complete medium containing 10% fetal bovine serum
(FBS). Cell lysates prepared at different times thereafter
were precipitated with antibody to Rb, and the resulting
immunoprecipitates were resolved on a denaturing gel,
transferred to nitrocellulose and blotted with the cognate
antibodies. The percentage of cells in S phase was estim-
ated by flow cytometric analysis of DNA content. As cells
approached S phase, Rb appeared to undergo phosphoryl-
ation, as manifested by its characteristic retardation in
electrophoretic mobility on denaturing gels (Figure 6A).
The kinetics of Rb phosphorylation and the rate of cell
cycle progression were quite similar in p21/p27 double-
null and wild-type MEFs. Hence, in the absence of the
CKIs, resident cyclin-dependent kinases are sufficient
to phosphorylate Rb, canceling its growth suppressive
function as cells exit G1phase.
Although the levels of cyclin D-dependent kinase
activity expressed in p21/p27 double-null cells were
reduced below the limits of detection (Figure 2), several
lines of evidence suggest that there may be some residual
kinase activity. First, using antibodies that detect Rb
phosphorylated on Ser780, a site reported to be specifically
phosphorylated by cyclin D-dependent kinases (Kitagawa
et al., 1996), forms of Rb phosphorylated on this residue
could be detected in cycling (lanes 4 and 6) but not in
quiescent (lane 5) p21/p27 double-null cells (Figure 6B).
CDK inhibitors promote cyclin D–CDK assembly
Fig. 6. Rb status and p16-induced arrest. (A) Wild-type and double-
null MEFs made quiescent by contact inhibition and serum starvation
were trypsinized, reseeded and stimulated to enter the cell cycle in
complete medium containing 10% FBS. Cell lysates prepared at
different times thereafter were immunoprecipitated with polyclonal
antibodies to mouse Rb, separated on denaturing gels and blotted with
monoclonal antibody to Rb. The position of the hypophosphorylated
form of Rb is indicated by the signal in starved cells, whereas
additional phosphorylation is connoted by the retardation in Rb’s
mobility as cells progress through the division cycle. The fraction of
cells in S phase was determined by flow cytometric analysis of DNA
content. Cells entered S phase by ~12 h and were predominately in
G2/M by the completion of the experiment. (B) Rb precipitated from
quiescent (lane 5) or proliferating (all other lanes) MEFs of the
indicated genotypes was immunoblotted with anti-Rb (lanes 1 and 2)
or with an antibody that specifically detects an epitope containing
pSer780 (lanes 3–6) (Kitagawa et al., 1996). The position of the
pSer780 form of Rb is indicated by arrows in the right margin.
(C) MEFs infected for 24 h with a control CD8 retrovirus or with
vectors encoding CD8 plus either p16INK4aor p27 were scored for
[3H]thymidine incorporation (2 h pulse). Results with the control CD8
vector were normalized to 100%. Standard errors (bars) were
calculated from several independent experiments.
Second, when we infected double-null cells with a retro-
virus encoding p16INK4a, we observed inhibition of S phase
entry, albeit not nearly to the same extent as that observed
with the wild-type MEFs (Figure 6C). As expected, both
MEF strains remained highly sensitive to growth arrest
by a retrovirus encoding p27. Since, unlike p27, INK4
proteins appear to specifically target CDK4 and CDK6,
these data imply that p21/p27 double-null cells are not
entirely devoid of cyclin D-dependent kinase activity.
We also measured total CDK2 or cyclin E-dependent
kinase activity in lysates of the various MEF strains, using
either histone H1 or GST-Rb as the substrate (Figure 7).
Although the levels of CDK2 were comparable in the
different cell strains (Figure 7D), both CDK2 and cyclin
E-dependent kinase activity were enhanced in p21/p27
double-null MEFs, as compared with those in wild-type
MEFs (Figure 7A–C). Therefore, in the absence of p21
and p27, unopposed CDK2 activity may compensate for
the severe reduction in CDK4 function. In agreement,
p21- and p27-null MEFs are not significantly perturbed
in cell cycle progression and exhibit generation times and
S phase fractions similar to those of wild-type cells (Figure
6A and data not shown).
Assembly of cyclin D–CDK complexes in tissues
from double-null mice
Although the above studies were performed with primary
MEFs, a clear prediction is that assembly of different
cyclin D-dependent kinases would be perturbed in many
other cell types. Extracts of whole liver from wild-type
and double-null mice expressed all three D-type cyclins
together with CDK6 (Figure 8, lanes 1 and 2). When these
were immunoprecipitated with a mixture of antibodies to
cyclins D1, D2 and D3 and then immunoblotted with
antibodies to CDK6, significantly fewer cyclin D–CDK6
complexes were observed in liver from double-null
animals. Similar results were obtained with CDK4 in
complexes with D1 (data not shown). T lymphocytes
primarily express cyclins D2 and D3 in conjunction with
CDK6 (Meyerson and Harlow, 1994), and thymic extracts
from double-null mice also contained much lower levels
of cyclin D–CDK6 complexes than those from wild-type
animals (Figure 8, lanes 3 and 4). Assembly of cyclin D–
CDK4 complexes was only modestly reduced in extracts
of kidney and heart from double-null mice (data not
shown). Therefore, despite the combinatorial nature of
expressed cyclin D–CDK complexes in different tissues
and the potential participation of other CKIs such as
p57Kip2in the assembly process, assembly of cyclin D-
dependent kinases was impaired in vivo.
CDK inhibitors of the Cip/Kip family, including p21Cip1,
p27Kip1and p57Kip2, negatively regulate cell cycle progres-
sion and enforce cell cycle arrest when expressed at high
levels (Elledge and Harper, 1994; Sherr and Roberts,
1995). Our results provide a different perspective in
showing that p21 and p27 are necessary for certain
processes that positively regulate cell cycle progression:
cyclin D assembly with CDK4, its stability and its nuclear
localization. Hence, the generally prevailing view of Cip/
Kip proteins as universal inhibitors of CDKs appears to
M.Cheng et al.
Fig. 7. CDK2 activities in MEFs lacking p21 and/or p27. Lysates from proliferating MEFs were precipitated with antibodies to CDK2 (A and B) or
to cyclin E (C), and immune complex kinase assays were performed using histone H1 (A and C) or GST-Rb (B) as substrates. All reactions were
stopped at the times indicated by heating samples to 85°C for 5 min in gel sample buffer containing SDS. Labeled proteins were resolved on
denaturing gels, which were dried and subjected to autoradiography. Excised slices containing substrates were rehydrated and radioactivity was
determined by liquid scintillation. Results are plotted in (A–C) for wild-type MEFs (squares), p21-null MEFs (triangles), p27-null MEFs (inverted
triangles) and double-null MEFs (diamonds). (D) CDK2 protein in MEFs of the indicated genotype was detected by immunoblotting with the
cognate antibody. Equal aliquots of lysate were separated per lane, and sites of antibody binding were detected by enhanced chemiluminescence.
portray too simple a picture of their regulatory effects on
the cell cycle. A more accurate representation is that these
CKIs are activators of CDK4 and inhibitors of CDK2.
This reformulation of the role of Cip/Kip proteins places
at the pivotal transition in the cell cycle between the
cyclin D-mediated responses to extrinsic mitogenic cues
and the CDK2-mediated progression from G1to S phase.
Stable association of p21 and p27 with active
cyclin D–CDK4 complexes in vivo
The solved structure of p27 in a complex with cyclin A–
CDK2 illustrates that a single p27 molecule can bind to
both the cyclin and CDK subunit and can disrupt the CDK
to dismantle its ATP binding site (Russo et al., 1996).
Although no analogous structure of a cyclin D–CDK–CKI
complex is yet available, p21 and p27 were found to be
potent inhibitors of binary cyclin D–CDK4 complexes
assembled from recombinant protein subunits (Harper
et al., 1993; Polyak et al., 1994; Toyoshima and Hunter,
1994). However, the idea that p21 and p27 differentially
regulate various cyclin–CDK complexes is consistent with
data that cyclin E–CDK2 and cyclin A–CDK2 are much
more susceptible to inhibition by these CKIs than are
cyclin D-dependent kinases in vivo (Soos et al., 1996;
Blain et al., 1997; LaBaer et al., 1997). As also observed
in our studies, enzymatically active cyclin D–CDK4 com-
plexes can be depleted from mammalian cell lysates with
antibodies to these CKIs. In turn, immune complexes
Fig. 8. Cyclin D–CDK assembly in mouse tissues. Tissue extracts
from liver and thymus of wild-type or p21/p27 double-null mice were
precipitated with a mixture of antibodies to cyclins D1, D2 and D3
(top panel) or anti-CDK6 (middle panel), and precipitated proteins
separated on denaturing gels were blotted with the same antibodies. In
parallel, cyclin D precipitates were blotted with anti-CDK6 (bottom) to
score for complex formation. Proteins were detected by enhanced
chemiluminescence using exposure times of 10 s (top, middle) and
60 s (bottom).
prepared in this manner retain Rb but not histone H1
kinase activity (Soos et al., 1996), a feature of substrate
specificity that distinguishes cyclin D-dependent holo-
enzymes from cyclin A-, B- and E-dependent kinases
(Matsushime et al., 1992). Therefore, p21 and p27 remain
CDK inhibitors promote cyclin D–CDK assembly
stably bound to active holoenzyme complexes during the
How can cyclin D–CDK complexes containing p21 and
p27 retain activity? One possibility is that higher order
cyclin D–CDK–CKI complexes recovered from mamma-
lian cells contain additional components that protect the
core binary enzyme from CKI-mediated inhibition (see,
for example, Zhang et al., 1994). Consistent with this
idea, the mass of active cyclin D–CDK6 complexes
recovered from T cells has been estimated at 150–170 kDa
(Mahony et al., 1998). Post-translational modifications of
the included subunits might also alter their activities. This
is not to say that Cip/Kip proteins cannot, under certain
circumstances, inhibit CDK4 and CDK6. For example, in
response to negative regulators of G1progression such as
cAMP or TGF-β, the accumulation of Cip/Kip proteins
can occlude cyclin D–CDK activation by CAK (Kato
et al., 1994; Polyak et al., 1994; Aprelikova et al., 1995).
Clearly, the nature of the active holoenzymes expressed
in mammalian cells needs to be better clarified.
The fact that Cip/Kip proteins enter into stable,
enzymatically active complexes with cyclin D and CDK4
subunits highlights a second, non-catalytic function of
cyclin D–CDKs: to ‘titrate’ p21 and p27, thereby freeing
other CDKs from their constraint. In the face of an
inhibitory threshold set by the CKIs, the latter process
sets a dependency of CDK2 activity on the mitogen-
stimulated assembly of cyclin D–CDK complexes, thereby
coordinating the sequential activities of these enzymes as
Cip/Kip regulation of cyclin D–CDK assembly
The loss of both p21 and p27 in MEF strains decreased
the steady-state levels of assembled cyclin D–CDK4
complexes ?10-fold and lowered cyclin D- and CDK4-
associated Rb kinase activities to undetectable levels. The
turnover of D-type cyclins was accelerated in cells lacking
both CKIs and their overall levels were diminished.
Nonetheless, the reduction in cyclin D levels per se did
not account for their failure to assemble with catalytic
subunits because ectopically overexpressed cyclin D1,
whether located primarily in the nucleus or cytoplasm,
was also unable to associate with CDK4 in this setting.
Conversely, reintroduction of p21 or p27 into double-
null cells reconstituted the formation of cyclin D–CDK
complexes and increased the stability of cyclin D1.
Assembly of D-type cyclins with CDK4 requires several
steps. Proper folding of CDK4 relies on the chaperone
function of a cytoplasmic complex that includes Hsp90/
Cdc37 (Dai et al., 1996; Stepanova et al., 1996). Once
released from this complex, CDK4 can either enter into
complexes with a D-type cyclin or can accumulate in
enzymatically inactive binary complexes with an INK4
protein. The fate of CDK4 is largely determined by the
availability of cyclin D subunits, which accumulate in
response to mitogenic signaling, but how p21 and p27
unclear. Both of the latter CKIs contain distinct binding
sites for CDKs and cyclins, which enable them to contact
both subunits simultaneously (Toyoshima and Hunter,
1994; Chen et al., 1995, 1996; Luo et al., 1995; Nakanishi
et al., 1995; Lin et al., 1996; Russo et al., 1996). Hence,
their binding to cyclin D and CDK4 might stabilize
complex formation. Interactions between p21/p27 and
CDK4/CDK6 might also prevent INK4 binding to the
catalytic subunits (Reynisdottir and Massague ´, 1997),
diverting CDK4 into cyclin D-containing complexes and
facilitating assembly through a less direct competitive
mechanism. Cells lacking p27 and p21 would be expected
to accumulate more CDK4–INK4 complexes, and under
these conditions, cyclin D1 should be destabilized (Bates
et al., 1994; Parry et al., 1995). Increased binding of
CDK4 to INK4 proteins was indeed observed, but its
association with Cdc37 was diminished relative to that in
wild-type cells. A significant fraction (~30%) of CDK4
remained unbound to INK4 proteins or to Cdc37 in
double-null MEFs, arguing that mechanisms other than
binding play a role in the assembly process.
Both the levels and assembly promoting activities of
p21 and p27 are governed by mitogenic signals. For
instance, p21 is frequently induced in cells entering the
cycle from a quiescent state, whereas p27 levels are
generally high in quiescent cells but fall prior to their
entry into S phase (Firpo et al., 1994; Kato et al., 1994;
Nourse et al., 1994). Hence, p27 should not be limiting
in promoting the association of cyclin D1 with CDK4 as
cells enter the cycle, but its presence in a quiescent cell
is still incapable of assisting assembly of cyclin–CDK
complexes (Matsushime et al., 1994). While it might be
argued that the level of cyclin D1 in quiescent cells is
simply too low to allow its entry into complexes with
CDK4, ectopically expressed D1 subunits are also unable
to assemble with CDK4 in serum-starved fibroblasts
(Matsushime et al., 1994) and require signals via the
Ras-Raf1-MEK-ERK kinase cascade to ensure complex
formation (Cheng et al., 1998). One scenario is that p27
or p21 is subject to phosphorylation by ERKs, and that
only the appropriately modified forms of the CKIs are
able to promote cyclin D–CDK complex formation. Both
of these CKIs are phosphoproteins, and p27 can be
phosphorylated on serine by ERK1 (Zhang et al., 1994;
Alessandrini et al., 1997). However, induction of
enzymatically active MEK1 in NIH 3T3 fibroblasts was
unaccompanied by detectable phosphorylation of p21 or
p27. Also, a mutant version of p27 in which the ERK1
phosphorylation site was changed to alanine was still able
to promote cyclin D1–CDK4 assembly (data not shown).
Other possibilities are that cyclin D and CDK4 must be
phosphorylated prior to assembly, or that the chaperone
activity of the Cdc37/hsp90 complex can be regulated
Several factors are likely to contribute to added com-
plexity in living animals. First, different cell types exhibit
varying levels of Cip/Kip proteins in vivo, so the extent
of assembly promoting activity attributed to each of these
CKIs likely varies between tissues. Double-null mice
continue to express cyclin D–CDK4 complexes, albeit at
reduced levels, in many tissues. Although p57Kip2seems
not to be required for assembly of cyclin D-dependent
kinases in cultured MEFs, it may well promote assembly
in other tissues. The possibility that new more distantly
related Cip/Kip family members may be identified and be
shown to play a role in cyclin–CDK assembly cannot be
formally excluded. Secondly, while MEFs preferentially
synthesize cyclin D1 and D2 in complexes with CDK4,
M.Cheng et al.
other cell types such as T lymphocytes, for example,
express D3 instead of D1 and much more CDK6 than
CDK4 (Ajchenbaum et al., 1993; Meyerson and Harlow,
1994). Therefore, the six cyclin D-dependent kinases
(containing D1, D2 or D3 with CDK4 or CDK6) are
likely to be differentially regulated by Cip/Kip family
were only modestly reduced in MEFs lacking p21 or p27,
as compared with those in cells lacking both CKIs.
Possibly, the loss of one of the CKIs facilitates compensa-
tion by the other. Finally, CKIs themselves are regulated
by signals that affect cell proliferation, organismal
development and cell differentiation (Elledge and Harper,
1994; Sherr and Roberts, 1995). As a singular example,
the Cip1 gene is directly regulated by p53 (El-Deiry et al.,
1993; Dulic et al., 1994), and in cancer cells containing
mutant p53, the levels of p21 are generally reduced
making other CKIs more likely to promote cyclin D–CDK
assembly under such circumstances.
CKIs affect cyclin D stability and nuclear
Cyclin D1 accumulates in the nucleus during G1phase
but redistributes into the cytoplasm during S phase (Baldin
et al., 1993). D-type cyclins and CDK4 lack obvious
nuclear localization signals (NLSs), and p21 or p27 can
promote the nuclear import of cyclin D1–CDK4 through
putative NLSs at their C-termini (LaBaer et al., 1997).
When cyclin D1 was ectopically overexpressed in wild-
type MEFs, most of it localized to the nucleus, but in
p21/p27 double-null cells, it was largely confined to the
cytoplasm. Therefore, under conditions in which cyclin
D1 levels are relatively high, the absence of p21 and p27
can limit its nuclear accumulation. However, assembly of
cyclin D–CDK complexes per se does not drive their
nuclear uptake, since endogenous cyclin D1 expressed at
much reduced levels in p21/p27 double-null cells was
able to accumulate in the nucleus, albeit less efficiently.
Moreover, a cyclin D1 (T156A) mutant that assembles
with CDK4 remains largely cytoplasmic, although its
nuclear entry can be enforced by p21 overexpression
(Diehl and Sherr, 1997). Conversely, the D1 (T286A)
mutant is preferentially retained in the nucleus even in
p21/p27 double-null cells where it does not undergo
assembly. Indeed, this underscores the fact that assembly
is inhibited in the absence of CKIs irrespective of whether
D1 is predominately cytoplasmic or nuclear. We conclude
that cyclin D–CDK assembly and nuclear uptake are
separable functions to which the CKIs contribute inde-
The overall levels of cyclins D1 and D2 were signific-
antly reduced in MEFs lacking both p21 and p27, but
cyclin D1 was stabilized and its levels were restored when
either CKI was reintroduced into the double-null cells.
How might the presence of CKIs affect cyclin D stability?
Phosphorylation of cyclin D1 on Thr286 targets it for
degradation, and elimination of this threonine markedly
increases the half-life of cyclin D1 in proliferating cells
(t1/2?3 h) (Diehl et al., 1997). Cyclins D2 and D3 appear
subject to similar controls (our unpublished data). One
possibility, then, is that binding of p21 or p27 to cyclin
D1–CDK4 complexes can partially suppress D1 phos-
phorylation on Thr286, thereby helping to stabilize the
cyclin. Phosphorylation of cyclin D1 on Thr286 is medi-
ated by GSK-3β, and although cyclin D1 turnover in
proliferating cells is relatively rapid (t1/2? 25 min),
decreased signaling through the Ras-PI3K-Akt pathway
activates GSK-3β and further shortens the half-life of
cyclin D1 to ~12 min (Diehl et al., 1998). Moreover,
overexpression of an active, but not kinase-defective form
of GSK-3β in mouse fibroblasts causes a redistribution of
cyclin D1 from the cell nucleus to the cytoplasm (Diehl
et al., 1998). Therefore, by suppressing Thr286 phos-
phorylation, CKIs might affect the stability of cyclin D1
via two mechanisms: by promoting its nuclear retention
and by preventing its targeting to proteasomes.
Consequences of Cip/Kip loss during the cell
Remarkably, MEFs lacking p21 and p27 did not exhibit
overtly aberrant cell cycles. Indeed, our failure to detect
cyclin D-dependent kinase activity in cells lacking both
p21 and p27 raised the possibility that these kinases are
not required for cell cycle progression. In an attempt to
test directly whether p21/p27 double-null cells lack all
cyclin D-dependent kinase activity, we infected these cells
with retroviruses encoding the CDK4- and CDK6-specific
inhibitor, p16INK4a. Proteins of the INK4 family are pre-
sumed to act specifically as inhibitors of cyclin D-depend-
ent kinases and are only able to arrest cells that retain Rb
function (Koh et al., 1995; Lukas et al., 1995; Medema
et al., 1995). Despite the absence of detectable cyclin D-
dependent kinase activity, p21/p27 double-null MEFs
retained some sensitivity to the cell cycle inhibitory effects
of overexpressed p16INK4a. Even more inhibition was
obtained using adenovirus vectors that programmed higher
levels of ectopic protein expression (data not shown). In
addition, Rb was phosphorylated on at least one site that
Therefore, cells lacking p21 and p27 might well express
cryptic cyclin D-dependent kinase activity. In short, while
we were unable to resolve whether cyclin D-dependent
kinases are dispensable for the division cycle in Rb-
positive cells lacking Cip/Kip proteins, it is evident that
such cells tolerate a significant reduction in enzyme
Although mice lacking cyclins D1 or D2 (or both)
exhibit focal developmental anomalies (Fantl et al., 1995;
Sicinski et al., 1995, 1996), two of the three D-type
cyclins are dispensable for most cell divisions in the life
of these animals. Indeed, if the key functions of cyclin
D-dependent kinases are to phosphorylate Rb and to titrate
CDK inhibitors, then their loss in p21/p27 double-null
cells should be well tolerated. In the absence of these
CKIs, the titration function of cyclin D-dependent kinases
would be superfluous, and unopposed cyclin E–CDK2
and cyclin A–CDK2 activities might be sufficient to
phosphorylate Rb. Based on changes in its electrophoretic
mobility, Rb phosphorylation increased as p21/p27-null
cells approached the G1/S boundary, and the kinetics of
cell cycle progression were remarkably similar to those
of wild-type MEFs. Together, our results argue that p21
and p27 positively regulate the assembly, stability and
of cyclin E–CDK2 and A–CDK2 are normally opposed
by these CKIs, but in their absence, the latter enzymes
CDK inhibitors promote cyclin D–CDK assembly
are likely to compensate for loss of cyclin D-dependent
Materials and methods
Cells and culture conditions
Mouse embryonic fibroblasts (MEFs) from animals deficient in p21Cip1,
p27Kip1or both CKIs were established as described previously (Zindy
et al., 1997) and maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% FBS, 2 mM glutamine, 0.1 mM non-
essential amino acids, 55 µM 2-mercaptoethanol and 100 U/ml each of
penicillin and streptomycin. To make them quiescent, confluent MEFs
were washed twice with phosphate-buffered saline (PBS) and cultured
in serum-depleted medium [DMEM with 0.1% FBS, 0.04% bovine
serum albumin (BSA), glutamine, penicillin and streptomycin] for 18 h.
Quiescent cells were trypsinized, re-plated at low density and stimulated
with complete medium containing 10% FBS to enter the division cycle,
and entry into S phase was monitored by estimating the DNA content
of propidium iodide-stained nuclei using fluorescence-activated flow
cytometry (Matsushime et al., 1991). The 293T retrovirus packaging
cell line and helper virus plasmid (Pear et al., 1993) were obtained from
C.Sawyers (University of California, Los Angeles) with permission from
David Baltimore (California Institute of Technology).
Immunoblotting, immunoprecipitation and
Immunoblotting of cyclin D1, cyclin D2, CDK4, CDK6, p21, p27 and
the detection of cyclin D–CDK complexes was performed as described
previously (Cheng et al., 1998). INK4 proteins were precipitated from
MEFs or mouse tissues as described (Zindy et al., 1997). For detection
of Rb protein, cells were disrupted in lysis buffer containing 50 mM
HEPES pH 7.0, 0.5% Nonidet P-40, 250 mM NaCl, 1 mM phenylmethyl-
sulfonyl fluoride (PMSF), 5 mM EDTA, 4 µg/ml aprotinin, 4 µg/ml
pepstatin (protease inhibitors from Sigma Chemicals, St Louis, MO),
0.5 mM sodium orthovanadate, 5 mM sodium fluoride, and 50 mM
β-glycerophosphate. Lysates were clarified by centrifugation, and protein
concentration was determined using a BCA assay kit (Pierce, Rockford,
IL). Protein (2 mg) was precipitated with rabbit antibody to Rb (SC-
050, Santa Cruz, CA) and collected with 30 µl of protein A–Sepharose
(Pharmacia Biotechnology, Uppsala, Sweden). After three washes in
lysis buffer, immune complexes resuspended in 30 µl gel sample buffer
[50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 1 mM dithiothreitol
(DTT), 0.1% Bromophenol Blue], were electrophoretically resolved on
denaturing polyacrylamide gels, transferred to nitrocellulose and probed
with the mouse monoclonal antibody to Rb (14001A, Pharmingen,
CA). Rb protein phosphorylated on Ser780 was detected by direct
immunoblotting using purified antibodies specifically reactive with a
phosphorylated epitope (Kitagawa et al., 1996). Metabolic labeling and
measurements of cyclin D1 turnover were performed as previously
described (Diehl et al., 1997). Indirect immunofluorescence was also
carried out according to previous methods (Diehl and Sherr, 1997)
except that the cells were fixed with 3.7% paraformaldehyde at room
temperature for 15–20 min and permeabilized with acetone for 5 min
Protein kinase assays
Immune complex kinase assays using 5 µg recombinant GST-Rb as
substrate were performed (Matsushime et al., 1994) using 500 µg total
lysate protein per reaction and immunoprecipitation with either rabbit
anti-peptide serum (RZ) directed to the CDK4 C-terminus or monoclonal
anti-cyclin D1 (D1-72-13G) derived previously (Matsushime et al.,
1994; Vallance et al., 1994). For immunodepletion, cell lysates were
first subjected to two rounds of immunoprecipitation with rabbit anti-
p21, rabbit anti-p27 or both, and proteins remaining in the supernatant
were precipitated with anti-CDK4 (serum RZ); the Rb kinase activity of
resulting CDK4 immune complexes was then determined as above. To
measure CDK2 activity, cells were lysed in 50 mM Tris–HCl pH 7.4,
250 mM NaCl, 0.1% Triton X-100, 10 mM β-glycerophosphate, 0.5 mM
sodium vanadate, 1 mM sodium fluoride, 1 mM DTT, 1 mM PMSF,
4 µg/ml aprotinin, and 4 µg/ml pepstatin. Clarified lysates (200 µg
protein per sample) were precipitated for 3 h at 4°C with antisera to
cyclin E (M-20) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or
CDK2 (sera RCCplus RDD) (Matsuoka et al., 1994) plus 20 µl protein
A–Sepharose. Immune complexes were washed twice with the same
lysis buffer and twice with kinase buffer (50 mM HEPES pH 8.0,
10 mM MgCl2, 1 mM DTT).Reaction mixtures (50 µl kinase buffer)
contained 30 µM ATP with 10 µCi [γ-32P]ATP plus 10 µg histone H1
or 5 µg GST-Rb. All reactions were stopped by adding 1/3 volume 3?
gel sample buffer and heating at 85°C for 5 min. Labeled proteins were
resolved on denaturing polyacrylamide gels, which were dried and
subjected to autoradiography.
Virus production and infection
Human kidney 293T cells were transfected (Chen and Okayama, 1987)
with 15 µg of ecotropic helper retrovirus plasmid plus 15 µg of SRα
vector DNA encoding p21, p27, p16INK4aor Flag-tagged cyclin D1
(WT) and cyclin D1 (T286A). Cell supernatants containing infectious
retroviral pseudotypes were harvested 24–60 h post-transfection, pooled
on ice and filtered (0.45 µm membrane). Virus infections of exponentially
growing MEFs were performed in a 9% CO2atmosphere with 5 ml of
virus-containing culture supernatants plus 10 µg/ml polybrene (Sigma,
St Louis, MO) for each 100 mm diameter culture dish. After 5 h, 10 ml
fresh medium was added, and medium was changed 24 h later. Cells
were harvested 48 h after infection, and the percentage of cells in S
phase was determined by flow cytometric analysis of DNA content
(Matsushime et al., 1991) or by incorporation of [3H]thymidine into
replicating cell DNA (Kamijo et al., 1997).
We thank Dr Yoichi Taya for generously supplying antibodies to the Rb
epitope containing phosphoserine 780, and Carol Bockhold, Erin Randell
and Esther Van der Kamp for excellent technical assistance. This work
was supported in part by NIH grant CA-56819 to M.F.R., by Cancer
Center Core Grant CA-21765 and by the American Lebanese Syrian
Associated Charities (ALSAC) of St Jude Children’s Research Hospital.
M.C. is supported by NIH training grant T32-CA-70089 and J.P.O. by
the Human Frontier Science Program. J.M.R. and C.J.S. are Investigators
of the Howard Hughes Medical Institute.
Independent regulation of human D-type cyclin gene expression during
G1phase in primary human T lymphocytes. J. Biol. Chem., 268,
Aktas,H., Cai,H. and Cooper,G.M. (1997) Ras links growth factor
signaling to the cell cycle machinery via regulation of cyclin D1 and
the cdk inhibitor p27Kip1. Mol. Cell. Biol., 17, 3850–3857.
Albanese,C., Johnson,J., Watanabe,G., Eklund,N., Vu,D., Arnold,A. and
Pestell,R.G. (1995) Transforming p21rasmutants and c-Ets-2 activate
the cyclin D1 promoter through distinguishable regions. J. Biol.
Chem., 270, 23589–23597.
Alessandrini,A., Chiaur,D.S. and Pagano,M. (1997) Regulation of the
cyclin-dependent kinase inhibitor
phosphorylation. Leukemia, 11, 342–345.
Aprelikova,O., Xiong,Y. and Liu,E.T. (1995) Both p16 and p21 families
of the cyclin-dependent kinase
phosphorylation of cyclin-dependent kinases by the CDK-activating
kinase. J. Biol. Chem., 270, 18195–18197.
Baldin,V., Lukas,J., Marcote,M.J., Pagano,M. and Draetta,G. (1993)
Cyclin D1 is a nuclear protein required for cell cycle progression in
G1. Genes Dev., 7, 812–821.
Bates,S., Parry,D., Bonetta,L., Vousden,K., Dickson,C. and Peters,G.
(1994) Absence of cyclin D/cdk complexes in cells lacking functional
retinoblastoma protein. Oncogene, 9, 1633–1640.
Blain,S.W., Montalvo,E. and Massague,J. (1997) Differential interaction
of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1with cyclin A–
Cdk2 and cyclin D2–Cdk4. J. Biol. Chem., 272, 25863–25872.
Boudewijn,M., Burgering,Th. and Coffer,P.J. (1995) Protein kinase
B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction.
Nature, 376, 599–602.
Brugarolas,J., Chandrasekaran,C., Gordon,J.I., Beach,D., Jacks,T. and
Hannon,G.J. (1995) Radiation-induced cell cycle arrest compromised
by p21 deficiency. Nature, 377, 552–557.
Chan,F.K.M., Zhang,J., Cheng,L., Shapiro,D.N. and Winoto,A. (1995)
Identification of human/mouse p19, a novel CDK4/CDK6 inhibitor
with homology to p16INK4. Mol. Cell. Biol., 15, 2682–2688.
Chen,C. and Okayama,H. (1987) High-efficiency transformation of
mammalian cells by plasmid DNA. Mol. Cell. Biol., 7, 2745–2752.
Ando,K., DeCaprio,J.A.and Griffin,J.D.(1993)
(CDK) inhibitors block the
M.Cheng et al.
Chen,J., Jackson,P.K., Kirschner,M.W. and Dutta,A. (1995) Separate
domains of p21 involved in the inhibition of cdk kinase and PCNA.
Nature, 374, 386–388.
Chen,J., Saha,P., Kornbluth,S., Dynlacht,B.D. and Dutta,A. (1996)
Cyclin-binding motifs are essential for the function of p21CIP1. Mol.
Cell. Biol., 16, 4673–4682.
Cheng,M., Sexl,V., Sherr,C.J. and Roussel,M.F. (1998) Assembly of
cyclin D-dependent kinase and titration of p27Kip1regulated by
mitogen-activated protein kinase kinase (MEK1). Proc. Natl Acad.
Sci. USA, 95, 1091–1096.
Cross,D.A.E., Alessi,D.R., Cohen,P., Andjelkovich,M. and Hemmings,
B.A. (1995) Inhibition of glycogen synthase kinase-3 by insulin
mediated by protein kinase B. Nature, 378, 785–789.
Dai,K., Kobayashi,R. and Beach,D. (1996) Physical interaction of
mammalian CDC37 with CDK4. J. Biol. Chem., 271, 22030–22034.
Deng,C., Zhang,P., Harper,J.W., Elledge,S.J. and Leder,P. (1995) Mice
lacking p21Cip1/Waf1undergo normal development, but are defective
in G1checkpoint control. Cell, 82, 675–684.
Diehl,J.A. and Sherr,C.J. (1997) A dominant-negative cyclin D1 mutant
prevents nuclear import of cyclin-dependent kinase-4 (CDK4) and its
phosphorylation by CDK-activating kinase. Mol. Cell. Biol., 17,
Diehl,J.A., Zindy,F. and Sherr,C.J. (1997) Inhibition of cyclin D1
phosphorylation on threonine-286 prevents its rapid degradation via
the ubiquitin-proteasome pathway. Genes Dev., 11, 957–972.
Diehl,J.A., Cheng,M., Roussel,M.F. and Sherr,C.J. (1998) Glycogen
Synthase Kinase-3β regulates cyclin D1 proteolysis and subcellular
localization. Genes Dev., 12, 3499–3511.
Dudek,H., Datta,S.R., Franke,T.F., Birnbaum,M.J., Yao,R., Cooper,G.M.,
Segal,R.A., Kaplan,D.A. and Greenberg,M.E. (1997) Regulation of
neuronal survival by the serine-threonine protein kinase Akt. Science,
Dulic,V., Kaufmann,W.K., Wilson,S.J., Tlsty,T.D., Lees,E., Harper,J.W.,
Elledge,S.J. and Reed,S.I. (1994) p53-Dependent inhibition of cyclin-
dependent kinase activities in human fibroblasts during radiation-
induced G1arrest. Cell, 76, 1013–1023.
El-Deiry,W.S. et al. (1993) WAF1, a potential mediator of p53 tumor
suppression. Cell, 75, 817–825.
Elledge,S.J. and Harper,J.W. (1994) CDK inhibitors: on the threshold of
check points and development. Curr. Opin. Cell Biol., 6, 847–852.
Fantl,V., Stamp,G., Andrews,A., Rosewell,I. and Dickson,C. (1995) Mice
lacking cyclin D1 are small and show defects in eye and mammary
gland development. Genes Dev., 9, 2364–2372.
Firpo,E.J., Koff,A., Solomon,M.J. and Roberts,J.M. (1994) Inactivation
of a cdk2 inhibitor during interleukin 2-induced proliferation of human
T lymphocytes. Mol. Cell. Biol., 14, 4889–4901.
Morrison,D.K., Kaplan,D.R. and Tsichlis,P.N. (1995) The protein
kinase encoded by the Akt proto-oncogene is a target of the PDGF-
activated phosphatidylinositol 3-kinase. Cell, 81, 727–736.
Franke,T.F., Kaplan,D.R., Cantley,L.C. and Toker,A. (1997) Direct
regulation of the Akt proto-oncogene product by phosphatidylinositol-
3,4-bisphosphate. Science, 275, 665–668.
Guan,K.-L., Jenkins,C.W., Li,Y., Nichols,M.A., Wu,X., O’Keefe,C.L.,
Matera,A.G. and Xiong,Y. (1994) Growth suppression by p18, a
p16INK4A/MTS1- and p15INK4B/MTS2-related CDK6 inhibitor, correlates
with wild-type pRb function. Genes Dev., 8, 2939–2952.
Hannon,G.J. and Beach,D. (1994) p15INK4Bis a potential effector of
TGFβ-induced cell cycle arrest. Nature, 371, 257–261.
Harper,J.W., Adami,G.R., Wei,N., Keyomarsi,K. and Elledge,S.J. (1993)
The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1
cyclin-dependent kinases. Cell, 75, 805–816.
Collaboration of G1 cyclins in the functional inactivation of the
retinoblastoma protein. Genes Dev., 8, 1759–1771.
Hirai,H., Roussel,M.F., Kato,J.-Y., Ashmun,R.A. and Sherr,C.J. (1995)
Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin
D-dependent kinases. Mol. Cell. Biol., 15, 2672–2681.
Ashmun,R.A., Grosveld,G. and Sherr,C.J. (1997) Tumor suppression
at the mouse INK4a locus mediated by the alternative reading frame
product p19ARF. Cell, 91, 649–659.
Kato,J., Matsuoka,M., Polyak,K., Massague ´,J. and Sherr,C.J. (1994)
of cyclin-dependent kinase-4 activation. Cell, 79, 487–496.
Kelly,B.L., Wolfe,K.G. and Roberts,J.M. (1998) Identification of a
the retinoblastoma protein. Proc. Natl Acad. Sci. USA, 95, 2535–2540.
Kerkhoff,E. and Rapp,U.R. (1997) Induction of cell proliferation in
quiescent NIH 3T3 cells by oncogenic c-Raf-1. Mol. Cell. Biol., 17,
Kitagawa,M. et al. (1996) The consensus motif for phosphorylation by
cyclin D1-Cdk4 is different from that for phosphorylation by cyclin
A/E-Cdk2. EMBO J., 15, 7060–7069.
activation of phosphatidylinositol 3-kinase is regulated by receptor
binding of SH2-domain-containing proteins which influence ras
activity. Mol. Cell. Biol., 16, 5905–5914.
Koh,J., Enders,G.H., Dynlacht,B.D. and Harlow,E. (1995) Tumour-
derived p16 alleles encoding proteins defective in cell cycle inhibition.
Nature, 375, 506–510.
Krude,T., Jackman,M., Pines,J. and Laskey,R.A. (1997) Cyclin/cdk-
dependent initiation of DNA replication in a human cell-free system.
Cell, 88, 109–119.
LaBaer,J., Garrett,M.D., Stevenson,L.F., Slingerland,J.M., Sandhu,C.,
Chou,H.S., Fattaey,A. and Harlow,E. (1997) New functional activities
for the p21 family of CDK inhibitors. Genes Dev., 11, 847–862.
Lavoie,J.N., L’Allemain,G., Brunet,A., Mu ¨ller,R. and Pouysse ´gur,J.
(1996) Cyclin D1 expression is regulated positively by the p42/
p44MAPKand negatively by the p38/HOGMAPKpathway. J. Biol.
Chem., 271, 20608–20616.
Lee,M.-H., Nikolic,M., Bapista,C.A., Lai,E., Tsai,L.-H. and Massague ´,J.
(1996) The brain-specific activator p35 allows Cdk5 to escape
inhibition by p27Kip1in neurons. Proc. Natl Acad. Sci. USA, 93,
Lin,J., Reichner,C., Wu,X. and Levine,A.J. (1996) Analysis of wild-type
and mutant p21WAF-1gene activities. Mol. Biol. Cell, 16, 1786–1793.
Lukas,J., Parry,D., Aagaard,L., Mann,D.J., Bartkova,J., Strauss,M.,
Peters,G. and Bartek,J. (1995) Retinoblastoma protein-dependent cell
cycle inhibition by the tumor suppressor p16. Nature, 375, 503–506.
Lundberg,A.S. and Weinberg,R.A. (1998) Functional inactivation of the
retinoblastoma protein requires sequential modification by at least two
distinct cyclin–CDK complexes. Mol. Cell. Biol., 18, 753–761.
Luo,Y., Hurwitz,J. and Massague ´,J. (1995) Cell cycle inhibition mediated
by functionally independent CDK and PCNA inhibitory domains in
p21CIP1. Nature, 375, 159–161.
Mahony,D., Parry,D.A. and Lees,E. (1998) Active cdk6 complexes are
predominantly nuclear and represent only a minority of the cdk6 in
T cells. Oncogene, 13, 603–611.
Matsuoka,M., Kato,J., Fisher,R.P., Morgan,D.O. and Sherr,C.J. (1994)
Activation of cyclin-dependent kinase-4 (CDK4) by mouse MO15-
associated kinase. Mol. Cell. Biol., 14, 7265–7275.
Matsushime,H., Roussel,M.F., Ashmun,R.A. and Sherr,C.J. (1991)
Colony-stimulating factor 1 regulates novel cyclins during the G1
phase of the cell cycle. Cell, 65, 701–713.
Matsushime,H., Ewen,M.E., Strom,D.K.,
Roussel,M.F. and Sherr,C.J. (1992) Identification and properties of an
atypical catalytic subunit (p34PSKJ3/CDK4) for mammalian D-type G1
cyclins. Cell, 71, 323–334.
Matsushime,H., Quelle,D.E., Shurtleff,S.A., Shibuya,M., Sherr,C.J. and
mammalian cells. Mol. Cell. Biol., 14, 2066–2076.
Medema,R.H., Herrera,R.E., Lam,F. and Weinberg,R.A. (1995) Growth
suppression by p16ink4requires functional retinoblastoma protein.
Proc. Natl Acad. Sci. USA, 92, 6289–6293.
Meyerson,M. and Harlow,E. (1994) Identification of a G1 kinase activity
for cdk6, a novel cyclin D partner. Mol. Cell. Biol., 14, 2077–2086.
Mittnacht,S., Lees,J.A., Desai,D.,
Weinberg,R.A. (1994) Distinct sub-populations of the retinoblastoma
protein show a distinct pattern of phosphorylation. EMBO J., 13,
Nakanishi,M., Robetorge,R.S., Adami,G.R., Pereira-Smith,O.M. and
Smith,J.R. (1995) Identification of the active region of the DNA
synthesis inhibitory gene p21Sdi1/CIP1/WAF1. EMBO J., 14, 555–563.
Nourse,J., Firpo,E., Flanagan,W.M., Coats,S., Polyak,K., Lee,M.H.,
Massague ´,J., Crabtree,G.R. and Roberts,J.M. (1994) Interleukin-2-
mediated elimination of p27Kip1cyclin-dependent kinase inhibitor
prevented by rapamycin. Nature, 372, 570–573.
Parry,D., Bates,S., Mann,D.J. and Peters,G. (1995) Lack of cyclin D-
Cdk complexes in Rb-negative cells correlates with high levels of
p16INK4/MTS1tumour suppressor gene product. EMBO J., 14, 503–511.
Harlow,E., Morgan,D.O. and
CDK inhibitors promote cyclin D–CDK assembly
Pear,W.S., NoPan,M.L., Scott,M.L. and Baltimore,D. (1993) Production
of high-titer helper-free retroviruses by transient transfection. Proc.
Natl Acad. Sci. USA, 90, 8392–8396.
Polyak,K., Lee,M.-H., Erdjument-Bromage,H., Koff,A., Roberts,J.M.,
Tempst,P. and Massague ´,J. (1994) Cloning of p27Kip1, a cyclin-
dependent kinase inhibitor and a potential mediator of extracellular
antimitogenic signals. Cell, 78, 59–66.
Reynisdo ´ttir,I. and Massague ´,J. (1997) The subcellular locations of
p15INK4band p27Kip1coordinate their inhibitory interactions with
cdk4 and cdk2. Genes Dev., 11, 492–503.
Russo,A.A., Jeffrey,P.D., Patten,A.K., Massague ´,J. and Pavletich,N.P.
(1996) Crystal structure of the p27Kip1 cyclin-dependent kinase
inhibitor bound to the cyclin A–cdk2 complex. Nature, 382, 325–331.
Serrano,M., Hannon,G.J. and Beach,D. (1993) A new regulatory motif
in cell cycle control causing specific inhibition of cyclin D/CDK4.
Nature, 366, 704–707.
Sheaff,R., Groudine,M., Gordon,M., Roberts,J. and Clurman,B. (1997)
Cyclin E-CDK2 is a regulator of p27Kip1. Genes Dev., 11, 1464–1478.
Sherr,C.J. (1996) Cancer cell cycles. Science, 274, 1672–1677.
Sherr,C.J. and Roberts,J.M. (1995) Inhibitors of mammalian G1cyclin-
dependent kinases. Genes Dev., 9, 1149–1163.
Sicinski,P. et al. (1995) Cyclin D1 provides a link between development
and oncogenesis in the retina and breast. Cell, 82, 621–630.
Sicinski,P. et al. (1996) Cyclin D2 is an FSH-responsive gene involved
in gonadal cell proliferation and oncogenesis. Nature, 384, 470–471.
Soos,T.J. et al. (1996) Formation of p27–CDK complexes during the
human mitotic cell cycle. Cell Growth Differ., 7, 135–146.
Stepanova,L., Leng,X., Parker,S.B. and Harper,J.W. (1996) Mammalian
p50Cdc37is a protein kinase-targeting subunit of Hsp90 that binds and
stabilizes Cdk4. Genes Dev., 10, 1491–1502.
Stillman,B. (1996) Cell cycle control of DNA replication. Science, 274,
Toyoshima,H. and Hunter,T. (1994) p27, a novel inhibitor of G1cyclin/
cdk protein kinase activity, is related to p21. Cell, 78, 67–74.
Strom,D.K. and Sherr,C.J.(1994)
mammalian D-type G1cyclins. Hybridoma, 13, 37–44.
Vanhaesebroeck,B., Leevers,S.J., Panayotou,G. and Waterfield,M. (1997)
Phosphoinositide 3-kinases: a conserved family of signal transducers.
Trends Biochem. Sci., 22, 267–272.
Vlach,J., Hennecke,S. and Amati,B. (1997) Phosphorylation-dependent
degradationofthe cyclin-dependentkinaseinhibitorp27Kip1. EMBOJ.,
Sustained activation of extracellular-signal-regulated kinase 1 (ERK1)
is required for the continued expression of cyclin D1 in G1phase.
Biochem. J., 326, 61–68.
Weinberg,R.A. (1995) The retinoblastoma protein and cell cycle control.
Cell, 81, 323–330.
Winston,J.T., Coats,S.R., Wang,Y.-Z. and Pledger,W.J. (1996) Regulation
of the cell cycle machinery by oncogenic ras. Oncogene, 12, 127–134.
Zhang,H., Hannon,G.J. and Beach,D. (1994) p21-containing cyclin
kinases exist in both active and inactive states. Genes Dev., 8,
Zindy,F., Quelle,D.E., Roussel,M.F. and Sherr,C.J. (1997) Expression of
the p16INK4atumor suppressor versus other INK4 family members
during mouse development and aging. Oncogene, 15, 203–211.
Received October 15, 1998; revised January 20, 1999;
accepted January 28, 1999