Previous Article | Next Article 
Molecular and Cellular Biology, March 1999, p. 1981-1989, Vol. 19, No. 3
Imperial Cancer Research Fund Laboratories,
London WC2A 3PX, United Kingdom
Received 30 July 1998/Returned for modification 17 September
1998/Accepted 19 November 1998
To investigate the mode of action of the
p16INK4a tumor suppressor protein, we have
established U2-OS cells in which the expression of
p16INK4a can be regulated by addition or
removal of isopropyl- In mammalian cells, the activity of
cyclin-dependent kinases (CDKs) is regulated in part by the expression
of specific inhibitory proteins, termed CDK inhibitors (CKIs)
(65). Although their precise roles have yet to be
established, the prevailing view is that specific CKIs may be
responsible for diverting cells out of the proliferative cycle to
facilitate terminal differentiation or to maintain quiescence
(14), and there is considerable interest in their potential
to act in tumor suppression (64). Based on primary sequence
comparisons, two families of CKIs, commonly referred to as the CIP/KIP
and the INK4 proteins, respectively, have been identified in mammalian
cells, (14, 65); as well as having distinct structural
characteristics (46, 59), it is clear that these families
have distinct modes of action (19, 20).
The CIP/KIP proteins, p21CIP1/WAF1/sdi1
p27KIP1, and p57KIP2
are grouped together largely because they share common sequence
motifs that mediate interaction between the CKI and cyclin-CDK
complexes (8, 35, 36, 43, 50, 53, 71). It is in fact
possible to demonstrate direct interactions between
p21CIP1 and p27KIP1 with
cyclins in the absence of a kinase subunit (8, 16, 17, 20, 36,
71), although this may be an intermediate step in the formation
of a ternary cyclin-CDK-CKI complex (25, 43). The
CIP/KIP proteins can bind to a variety of cyclins and CDKs and in
assays based on expressing active complexes in insect cells, using
baculovirus vectors, it is clear that all three are capable of
inhibiting the kinase activity of CDK4 and CDK6 complexed to D cyclins
and of CDK2 complexed to either cyclin E or cyclin A (24, 25, 35,
43, 53, 71, 76). They therefore have the potential to block cell
cycle progression at multiple points, but the situation is complicated
by the fact that CIP/KIP proteins can also participate in active kinase
complexes, both in vivo and in vitro (5, 8, 16, 25, 33, 66,
78). This may reflect their ability to serve as assembly factors
in some circumstances, so that at low concentrations they may
facilitate the association of cyclins and CDKs whereas at higher
concentrations they function as inhibitors (33, 78). There
are also indications that p27KIP1, for example,
is a more potent inhibitor of CDK2 than CDK4 (5), suggesting
that there are important subtleties to these interactions that have yet
to be explored.
In contrast to the CIP/KIP family, the INK4 proteins,
p16INK4a, p15INK4b,
p18INK4c, and p19INK4d,
are highly related, each comprising between three and five ankyrin-type repeats with minimal amino- and carboxy-terminal extensions (reviewed in reference 58). All four INK4 proteins bind
directly to CDK4 and CDK6, and there is no evidence that they associate
with other CDKs (7, 18, 19, 21, 28, 29, 52, 62). They
therefore serve as specific inhibitors of cyclin D-dependent kinases
and have the capacity to induce a G1 cell cycle arrest by
preventing the phosphorylation of the retinoblastoma gene product, pRb
(18, 28, 32, 40, 47, 51, 61, 63, 69). The prototypic and
most illustrious member of the family, p16INK4a,
is a bona fide tumor suppressor that is incapacitated by homozygous deletion, mutation, or promoter methylation in a wide variety of
sporadic human tumors (58). Germ line mutations in the
p16INK4a gene are also associated with familial
melanoma (58).
In trying to determine why it is p16INK4a,
rather than other members of the INK4 family, that acts as a tumor
suppressor, we and others have noted that
p16INK4a levels increase significantly as
primary cells reach the end of their finite life span in culture
(2, 22, 37, 57, 75). This would be consistent with a role
for p16INK4a in establishing the G1
arrest associated with replicative senescence. Indeed, ectopic
expression of p16INK4a in primary fibroblasts
and some established cell lines elicits many of the phenotypic
characteristics associated with senescence (45, 72, 74). A
need to escape senescence would therefore provide a strong selection
against p16INK4a function during the
establishment of an immortal cell clone.
However, the mechanisms through which the INK4 family induce a cell
cycle arrest are still a matter of debate. Most of the evidence favors
the competitive binding model in which the direct association of INK4
proteins with CDK4 or CDK6 prevents them from interacting with their
regulatory D cyclins. As well as supporting in vitro data (19,
52), this mode of action has the logical appeal that in vivo, the
relatively unstable D-type cyclins bound to CDK4/CDK6 would be
progressively replaced by the more stable INK4 proteins
(52). This would explain why INK4 proteins are rarely found
in immune complexes with the D cyclins (19, 20, 28, 52, 60).
However, the opposing model holds that in some circumstances it is
indeed possible to find ternary complexes of INK4 proteins, D cyclins,
and CDKs. The strongest evidence is that recombinant INK4
proteins are capable of inhibiting the kinase activity of cyclin D-CDK
complexes assembled by using baculovirus systems without quantitatively
disrupting these complexes (28, 55). Similarly, in
mammalian cells arrested by induced expression of
p15INK4b or p19INK4d, the
INK4 proteins have been reported to coprecipitate with D cyclins and to
invoke a cell cycle arrest without displacing CDK4/CDK6 from the cyclin
D complexes (1, 55).
Because of these conflicting views, and the importance of
p16INK4a as a senescence regulator and tumor
suppressor, we have investigated what happens to cyclin-CDK-CKI
complexes in cells engineered for inducible expression of
p16INK4a. Most of the previous studies have
described the results simply in terms of binary and ternary
associations, with little focus on the stoichiometry of the complexes
or the proportions of the individual components involved in each
interaction. By using gel filtration chromatography to distinguish
complexes of different size, we show that the G1
arrest imposed by p16INK4a is accompanied
by a reassortment of components. By sequestering CDK4 and CDK6 in
inactive binary complexes, p16INK4a allows CDK2
to associate with cyclin D1 and releases enough
p27KIP1 to inhibit cyclin E-CDK2. The data
effectively eliminate the possibility that
p16INK4a forms stable ternary or higher-order
complexes with D-type cyclins.
Inducible cell lines.
The human osteosarcoma cell line U2-OS
was maintained at 37°C in Dulbecco modified Eagle's medium
supplemented with 10% fetal calf serum.
Isopropyl- Antibodies.
Immunoblotting of the D cyclins was done with
monoclonal antibodies DCS6 for cyclin D1, DCS3.1 for cyclin D2, and
DCS22 for cyclin D3 (3, 39). Corresponding
immunoprecipitations used the polyclonal antibody 287.3 for cyclin D1
(4) and the monoclonal antibodies DCS5.2 and DCS28,
respectively, for cyclins D2 and D3. Monoclonal antibodies HE12 and
HE172, used for Western blot detection and immunoprecipitation of
cyclin E, were provided by E. Lees.
p16INK4a was detected with monoclonal antibody
JC8 supplied by J. Koh, and pRb was detected with the
Pharmingen monoclonal antibody 14001A. pRb phosphorylated at S780 was
detected with a polyclonal antibody provided by Y. Taya
(31). The following polyclonal antibodies from Santa Cruz
were used for immunoprecipitation and/or Western blotting: CDK2
(sc-163), CDK4 (sc-601), p21CIP1 (sc-397),
p27KIP1 (sc-528), and cyclin A (sc-751).
Immunoprecipitation and immunoblotting.
Cells were recovered
from the culture flasks by treatment with trypsin and washed once with
phosphate-buffered saline by centrifugation and resuspension. Cells
were then lysed by resuspension in Tween lysis buffer (50 mM HEPES [pH
8.0], 10 mM MgCl2, 0.1% Tween 20, 1 mM dithiothreitol, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 5 µg of aprotinin
per ml, 100 µg of phenylmethylsulfonyl fluoride per ml) followed by
sonication for two 5-s pulses. Lysates were clarified by centrifugation
at 10,000 × g for 10 min, and the protein
concentrations were determined by using the Pierce bicinchoninic acid
protein assay reagents according to the manufacturer's instructions.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Induced Expression of
p16INK4a Inhibits Both CDK4- and CDK2-Associated
Kinase Activity by Reassortment of Cyclin-CDK-Inhibitor
Complexes
and
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-D-thiogalactopyranoside. As
expected, induction of p16INK4a results in a
G1 cell cycle arrest by inhibiting phosphorylation of the
retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and
CDK6. However, induction of p16INK4a also
causes marked inhibition of CDK2 activity. In the case of cyclin
E-CDK2, this is brought about by reassortment of cyclin, CDK, and
CDK-inhibitor complexes, particularly those involving p27KIP1. Size fractionation of the cellular
lysates reveals that a substantial proportion of CDK4 participates in
active kinase complexes of around 200 kDa. Upon induction of
p16INK4a, this complex is partly dissociated,
and the majority of CDK4 is found in lower-molecular-weight fractions
consistent with the formation of a binary complex with
p16INK4a. Sequestration of CDK4 by
p16INK4a allows cyclin D1 to associate
increasingly with CDK2, without affecting its interactions with the
CIP/KIP inhibitors. Thus, upon the induction of
p16INK4a, p27KIP1
appears to switch its allegiance from CDK4 to CDK2, and the
accompanying reassortment of components leads to the inhibition of
cyclin E-CDK2 by p27KIP1 and
p21CIP1. Significantly,
p16INK4a itself does not appear to form
higher-order complexes, and the overwhelming majority remains
either free or forms binary associations with CDK4 and CDK6.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-D-thiogalactopyranoside (IPTG)-regulatable p16INK4a was obtained by using the Stratagene
LAC-SWITCH system. The DNA fragment encoding
p16INK4a was excised from a pcDNA3-based plasmid
by using XbaI and BamHI and subcloned into
pBluescript to exploit the NotI sites in the polylinker. The
insert was then recovered by digestion with NotI and
subcloned into the NotI site in pOPRSVI. The orientation of the insert was determined by DNA sequencing. U2-OS cells were cotransfected with a 1:1 mixture of p16INK4a in
pOPRSVI and the regulatory plasmid p3'SS. Stable transfectants were
selected in hygromycin (150 µg/ml; Sigma) and geneticin (300 µg/ml;
Gibco) followed by the isolation of single colonies. Of 30 clones
tested, two (designated EH1 and EH2) were found to express p16INK4a under the control of the lac
promoter. EH1 cells were maintained under selective conditions, but to
ensure that the cells were not metabolically challenged during various
assays, the antibiotics were removed from the medium 24 h prior to
initiation of experiments. To induce expression, EH1 cells were split
at a ratio of 1:2 and 1 mM IPTG was added to the fresh medium.
Analysis of 35S-labeled proteins. To metabolically label the cellular proteins, EH1 cells (approximately 50% confluent) were incubated for 30 min in Dulbecco modified Eagle's medium lacking methionine and cysteine and supplemented with 10% dialyzed fetal calf serum. The cells were then labeled for 2 h in the same medium containing 50 µCi of [35S]methionine-cysteine (Promix; Amersham plc) per ml. Cell lysates were prepared by using Nonidet P-40 lysis buffer and processed as described previously (4). Labeled proteins were detected by autoradiography.
Sequential immunoprecipitations. To determine the proportion of p27KIP1 associated with different cyclins and CDKs, sequential immunoprecipitations were performed with antibodies covalently coupled to agarose beads. Typically, 450 µg of lysate was subjected to two rounds of precipitation with 30 µl of beads (50% slurry) at 4°C for 2 h. After each incubation, the beads were pelleted and the supernatants were transferred to the next set of beads. Each precipitate was then subjected to SDS-PAGE and immunoblotted for p27KIP1. Mock immunoprecipitations were carried out in parallel with anti-rabbit immunoglobulin G bound to beads (Sigma), and the supernatant from this control set of precipitations served as the control for the total amount of p27KIP1 present in the lysate.
Gel filtration chromatography. Gel filtration chromatography was carried out by using a Superdex 200 HR 10/30 column (Pharmacia) with a fast protein liquid chromatography system (BioLogic system; Bio-Rad). Samples of 4 mg of cell lysate in 250 µl of Tween lysis buffer were loaded onto the column and separated at a flow rate of 0.4 ml per min. The molecular mass standards (Sigma) used to calibrate the column were thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), myoglobin (17.6 kDa), and cytochrome c (12.4 kDa). Lysates from untreated and IPTG-treated cells were analyzed sequentially on the same column followed by a subset of molecular mass standards to ensure that the efficiency of separation and calibration of the column was being maintained.
CDK assays.
Kinase assays were carried out essentially as
described by Matsushime et al. (44) except that the
substrate was a glutathione S-transferase (GST) fusion
protein containing only the carboxy-terminal region of pRb
(48). Lysates from untreated and IPTG-treated EH1 cells were
immunoprecipitated with antisera to CDK4, CDK6, or CDK2 as described
above except that the incubation time was reduced to 5 h. Immune
complexes were washed three times with Tween lysis buffer and twice
with kinase reaction buffer (50 mM HEPES, pH 8.0, 10 mM
MgCl2, 2.5 mM EGTA, 1 mM dithiothreitol, 25 mM ATP); 25 µl of kinase assay buffer containing 2 µg of GST-Rb and 10 µCi of
[
-32P]ATP was added to each sample, and after
incubation at 30°C for 20 min, reactions were stopped by adding 25 µl of 2× dissociation buffer (23). Samples were boiled
for 2 min and resolved by SDS-PAGE in a 10% gel. The phosphorylated
substrate was detected initially by autoradiography and quantitated by
PhosphorImager (Molecular Dynamics) analysis using Imagequant software.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Cell cycle arrest by inducible p16INK4a.
To
examine the effect of p16INK4a on G1
CDK activity and investigate its mode of action, stable cell clones
that express p16INK4a under the control of an
IPTG-inducible promoter were derived. The osteosarcoma cell line U2-OS
was chosen as the recipient because it retains functional p53 and pRb
(11) and does not express endogenous
p16INK4a due to methylation of the exon 1
promoter (70). The clone selected for these studies, EH1,
showed a low basal level of p16INK4a
expression in the absence of IPTG, reflecting leakiness in the regulatable system (Fig. 1A).
However, the levels were comparable to the low amounts of
p16INK4a expressed in early-passage human
diploid fibroblasts and had no discernible effect on the growth
of the cells (data not shown). Upon addition of IPTG, the expression
of p16INK4a increased in a dose-dependent
manner, reaching a maximum level at around 1 mM IPTG (70).
Under these conditions, the cells expressed
p16INK4a at levels equivalent to those in
senescent human fibroblasts (Fig. 1A and data not shown).
|
-galactosidase activity (12, 45).
We next asked whether the induction of p16INK4a
had altered the steady-state levels of other cell cycle components that
might have contributed to the G1 arrest. Compared to the
marked change in p16INK4a expression, no
significant changes were observed in the levels of the CKIs
p21CIP1 and p27KIP1 (Fig.
1C). Likewise, the steady-state levels of cyclins D1, D2, and E were
unaffected, but substantially lower amounts of cyclins D3 and A were
detected in the IPTG-treated cells (Fig. 1C). No significant changes
were observed in CDK4 and CDK6 levels, but there was a modest down
regulation of CDK2 (Fig. 1C). The reduction was more striking for the
active, phosphorylated form of CDK2 (faster-migrating band) so that in
the IPTG-treated cells, CDK2 appeared to be primarily in its inactive,
unphosphorylated state (slower-migrating band).
Inhibition of CDK-associated kinase activity by inducible p16INK4a. Since p16INK4a binds directly to CDK4 and CDK6, and not to CDK2 (21, 52, 62), induction of p16INK4a in EH1 cells should principally affect the kinase activity associated with cyclin D-CDK complexes. CDK4 immunoprecipitates from EH1 cells, before and after IPTG treatment, were therefore tested for the ability to phosphorylate the carboxy-terminal domain of pRb in vitro. The specificity of the signal was confirmed by blocking the precipitation of CDK4 with an excess of the cognate peptide antigen. Upon induction of p16INK4a with IPTG, there was a reduction of the CDK4 activity to almost background levels (Fig. 2A).
|
Rearrangement of cyclin-CDK complexes upon expression of p16INK4a. Since p16INK4a does not bind directly to CDK2, we were interested in exploring the mechanisms behind its impact on CDK2 activity. In the case of cyclin A, the most likely explanation would be the reduced expression of the cyclin (Fig. 1C), but since cyclin E levels remained constant in IPTG-treated cells, the reduced kinase activity presumably occurred through a change in its association with other proteins, such as CIP/KIP type CKIs (55, 56). To address this possibility, selected cyclins and CKIs were immunoprecipitated from EH1 cells before and after p16INK4a induction, and the associated CDKs were detected by immunoblotting (Fig. 3A). As expected, although the total levels of CDK4 remained the same (lanes 1 and 2), there was significantly less CDK4 associated with cyclin D1 and significantly more CDK4 associated with p16INK4a in cells treated with IPTG. This would be consistent with the down regulation of CDK4-associated kinase activity. The redistribution of CDK4 was also apparent in the p27KIP1 immunoprecipitates. Thus, in untreated EH1 cells, CDK4 and p27 participated in a complex, presumably including D cyclins, that appeared to be substantially disrupted upon the induction of p16INK4a. Although a similar trend was apparent for complexes containing p21CIP1, the effects were less pronounced (Fig. 3A).
|
) IPTG
were immunoprecipitated with antisera against cyclin D1, cyclin E,
p16INK4a, p21CIP1, and
p27KIP1 as indicated. After fractionation by
SDS-PAGE, the precipitated proteins were immunoblotted for CDK4 (upper
panels) and CDK2 (lower panels). The total amounts of CDK4 and CDK2
were monitored by direct immunoblotting of a portion of the cell
lysates (Lysate). (B) Levels of p27KIP1 or
p21CIP1 associated with cyclin D1 or cyclin E
were determined as for panel A. (C) Lysates from untreated or
IPTG-treated cells were subjected to sequential immunoprecipitation
with antibodies against cyclins D1 plus D2, cyclin D3, cyclin E, and
cyclin A as indicated. Each antibody was covalently coupled to
agarose beads so that the supernatant from each immunoprecipitation
could be quantitatively recovered and used in the subsequent
precipitation. Two rounds of precipitation were conducted with each
antibody. The final supernatant (Residual p27) was then precipitated
with antiserum against p27KIP1. As a control, a
sample of the starting lysate was subjected to repeated cycles of
precipitation with nonimmune serum (Total p27). The amounts of
p27KIP1 in the various precipitates were then
compared by immunoblotting.
Size fractionation of cyclin-CDK-CKI complexes. One of the limitations of this simple coprecipitation approach is that it does not provide information about the stoichiometry of the various multicomponent complexes. To try to address this question, protein lysates from IPTG-treated or untreated EH1 cells were subjected to gel filtration to separate complexes of different sizes. The fractionated lysates were first analyzed directly by immunoblotting to determine the proportions of each protein in different size fractions (Fig. 4). In asynchronously proliferating EH1 cells, p21CIP1 and p27KIP1 were found exclusively in high-molecular-mass complexes of around 200 kDa, with the p21CIP1 complex consistently appearing marginally larger than the p27KIP1 complex. These complexes appeared unchanged when p16INK4a expression was induced with IPTG. Interestingly, the induced p16INK4a was distributed between two major peaks, the smaller of which would be consistent with free p16INK4a. The larger peak, running at approximately 50 kDa, would be compatible with binary complexes between p16INK4a and either CDK4 or CDK6, but not with the formation of ternary complexes incorporating a D cyclin. However, since this main peak tailed into higher-molecular-mass fractions, it remains possible that a small percentage of the induced p16INK4a entered complexes containing both cyclins and CDKs (see below).
|
Changes in composition of CDK complexes upon induction of
p16INK4a.
Direct immunoblotting provided
information about the distribution of cell cycle components in
complexes of different sizes but not about the composition or kinase
activity of the various complexes. Individual fractions were therefore
immunoprecipitated with antibodies against either CDK4 or CDK2, and the
associated cyclins and CDK inhibitors were visualized by
immunoblotting. As well as separating equal amounts of lysates by gel
filtration, care was taken to analyze the immunoblots from IPTG-treated
and untreated cells under identical conditions (simultaneously) in order to assess quantitative differences in the proteins bound to the
CDKs. The CDK4 and CDK2 immunoprecipitates from untreated cells were
also assayed for kinase activity on pRb as the substrate. The
immunoprecipitated CDK4 recapitulated the distributions observed in
Fig. 4, with three major peaks at 
450, 200, and 50 kDa (Fig. 5A). Kinase activity was predominantly
associated with the 200-kDa peak, corresponding with peaks in the
distribution of D cyclins, p21CIP1, and
p27KIP1 that coprecipitated with CDK4.
|
|
Time course of p16INK4a-induced changes. Although we found no evidence for ternary complexes at 48 h postinduction, it remained formally possible that a transient association of p16INK4a with cyclin D1 and CDK4 was responsible for the inhibition of CDK4-associated kinase activity and G1 arrest. To explore this possibility, we prepared lysates from EH1 cells at various times after addition of IPTG and examined the association of CDK4 with either cyclin D1 or p16INK4a by immunoprecipitation and Western blotting. Upon addition of IPTG, the steady-state levels of p16INK4a increased over the first 6 to 8 h and then remained relatively constant (Fig. 7A and additional data not shown). This was paralleled by an increase in the association of CDK4 with p16INK4a as assayed by immunoprecipitation of either CDK4 or p16INK4a (Fig. 7A). However, no cyclin D1 was detected in the p16INK4a immunoprecipitates at any time point, and vice versa (negative data not shown).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
The induction of p16INK4a in EH1 cells had a number of striking effects on the composition of cyclin-CDK complexes, the combined result of which was a G1 arrest. A major aim of this work was to determine whether the primary effect of p16INK4a was to divert CDK4 from active complexes, as predicted by the competitive binding model, or to inhibit preexisting cyclin D-CDK4 by joining ternary or higher-order complexes. In this respect, the gel filtration results effectively preclude the latter model since the overwhelming majority of p16INK4a in IPTG-treated EH1 cells was found in fractions in the 50- and 15- to 20-kDa size ranges. Selective immunoprecipitation and Western blotting confirmed that these peaks correspond to CDK-bound and free p16INK4a, respectively, and attempts to find cyclin D1 and p16INK4a in the same fractions were consistently negative. Although we cannot formally exclude the transient formation of higher-order complexes, we estimate that in a steady-state situation, 48 h postinduction, less that 1% of the total p16INK4a in the cells eluted in complexes larger than 50 kDa.
Previous studies using similar inducible expression systems demonstrated coprecipitation of D cyclins and either p15INK4b or p19INK4d but did not address the proportions of the INK4 proteins in such complexes (1, 55). While it is possible that different INK4 proteins have different properties in this regard, the structural data suggest that they adopt similar three-dimensional conformations (6, 38, 46, 73). Another possibility is that the rapid induction of artificially high levels of INK4 protein leads to a transient association with cyclin-CDK complexes that is rarely seen under physiological conditions. In the previous studies, the cells were harvested relatively soon after induction (1, 55). However, we found no evidence for coprecipitation of cyclin D1 and p16INK4a during a time course of p16INK4a induction (Fig. 7 and data not shown). On the contrary, the interactions of newly synthesized components observed at various time points would be entirely consistent with a competitive binding model in which the unstable cyclin is gradually replaced by the more stable inhibitor. The rate at which the different complexes appear to change would of course depend on the stoichiometry of the different components.
Another feature of our data that contrasts with conclusions drawn elsewhere (55) is that the induction of p16INK4a demonstrably affected the composition of cyclin D-CDK complexes. In this respect, our data are more in line with those reported by Adachi et al. and Sandhu et al., both of whom showed a reduction in cyclin D-CDK4 interactions when INK4 levels were elevated (1, 60). We are also in complete agreement with a recent report by Jiang et al. based on inducible expression of p16INK4a (30). Thus, in IPTG-treated EH1 cells a substantial proportion of the CDK4 relinquished its association with cyclin D1 to form complexes with p16INK4a. One consequence was the broadening of the cyclin D1 peak on the gel filtration column, and it will be interesting to determine whether the smaller cyclin D1 complexes, at around 120 to 150 kDa, contain any kinase subunits. However, much of the cyclin D1 remains in the 200-kDa peak and the total amounts in the cell are unchanged, ruling out the increased turnover of cyclin D1 when it is dissociated from CDK4. A likely reason is that the CDK4 is replaced in the complex by CDK2.
Cyclin D1 has been previously shown to interact with CDK2 in some cell types (4, 77), and this association is increased in senescent fibroblasts (13). However, it is predominantly the unphosphorylated, inactive form of CDK2 that coprecipitates with cyclin D1, consistent with the change in the ratio of the two forms when p16INK4a is induced in EH1 cells. It has been formally demonstrated that cyclin D1-CDK2 complexes are inactive as kinases and insensitive to activation by cyclin H-CDK7 (27), but the situation is less clear for cyclins D2 and D3; coexpression with CDK2 in baculovirus-based systems has been shown to produce active kinases (15).
Replacement of CDK4 by CDK2 puts a different complexion on the reassortment of the various components in IPTG-treated EH1 cells. It has been previously surmised that induction of INK4 proteins causes p27KIP1 to move off cyclin D1-CDK4 complexes and onto cyclin E-CDK2 (30, 55, 56, 60), but our data show little if any displacement of p27KIP1 from cyclin D1. A more likely interpretation, therefore, is that p27KIP1 remains associated with cyclin D1, to which it can bind directly in the absence of a CDK (8, 16, 17, 20, 36, 71). The substitution of CDK2 for CDK4 in this complex would then explain the dramatic shift in allegiance of p27KIP1 from CDK4 to CDK2 shown in Fig. 3 and elsewhere (30, 55, 56, 60). The one facet of the data not explained by this model is the increase in the amount of p27KIP1 and p21CIP1 bound to cyclin E after p16INK4a induction (Fig. 3 and reference 30). Several possibilities can be considered. The first is that the levels of cyclin E are generally much lower than those of cyclin D1 so that a severalfold increase in the amount of p27KIP1 that coprecipitates with cyclin E may represent a very small proportion of the amount normally bound to cyclin D1. Another is that there is an appreciable reduction in the expression of cyclin D3 in IPTG-treated EH1 cells which, together with the inability to activate cyclin A expression, leads to a surplus of p27KIP1 that accumulates on cyclin E-CDK2 complexes. The immunodepletion experiments shown in Fig. 3C favor the latter interpretation since there is a substantial reduction in cyclin D3-p27KIP1 complexes in the arrested cells. What causes this reduction remains a matter of conjecture, but one possibility would be that, at least in some cell systems, cyclin D3 expression is delayed relative to that of cyclins D1 and D2. Cyclin D3 expression may therefore be partially dependent on the activation of CDK4 or CDK6.
The net effect of these changes is a marked reduction in the kinase activity associated with CDK2, whether it is bound to cyclin E or to cyclin A. However, it remains unclear whether this inhibition of CDK2 activity is an essential factor in the cell cycle arrest imposed by p16INK4a or is simply an adjunct to the direct inhibition of cyclin D-associated CDK4 and CDK6. Our data are clearly at odds with a previous report that cotransfection of p16INK4a can prevent the phosphorylation of pRb without inhibiting the activity of cyclin E-CDK2 (41) but agree with those recorded for inducible or ectopic expression of p15INK4b (55, 56) and p16INK4a (30). The IPTG-treated EH1 cells arrest with pRb in its hypophosphorylated state, consistent with the observed inhibition of CDK2, -4, and -6, but this would also be consistent with models in which the hyperphosphorylation of pRb by cyclin E-CDK2 is dependent on prior phosphorylation by cyclin D-dependent kinases (26, 41). Nevertheless, it is clear from Fig. 2 that upon p16INK4a-mediated arrest, pRb is not phosphorylated on Ser780, a site specifically targeted by cyclin D-dependent kinases (31).
It is now well established that cyclins and CDKs are found in multicomponent complexes within mammalian cells, although the exact composition of these complexes is not fully understood (10, 42, 49, 54, 67). Most cyclins show a major peak of around 150 to 220 kDa, although size estimates vary in different studies, and our data are in broad agreement. CDK4 and CDK6 are found primarily in three distinct complexes of very different sizes. As recently reported for CDK6 (42), a significant proportion of the CDK4 eluted with an apparent molecular mass in excess of 400 kDa. The other components of this complex have not been identified but may include the molecular chaperones HSP90 and CDC37 (9, 34, 42, 68). In the case of CDK6, Mahony et al. noted that the minor peak at 170 to 200 kDa corresponded to active kinase (42), and our data for EH1 cells concur (not shown). However, a much higher proportion of the CDK4 is present in the active kinase complex at the 170- to 200-kDa peak, and in contrast to CDK6, only a minor fraction of the CDK4 is found in binary complexes with INK4 proteins at ~50 kDa. Since current methods do not allow quantitative comparisons of the CDK4- and CDK6-associated kinase activity, it is not possible to assess the relative contributions of the two kinases to G1 progression. Nevertheless, our data indicate that both active complexes are affected by the induction of p16INK4a.
It is clear that the interactions between cell cycle regulatory components are integrally connected. By making one alteration, such as increasing the levels of p16INK4a, we find that several multicomponent complexes undergo reassortment and that the impact of the inhibitor is more pleiotropic than simply altering the activity of the proteins to which it directly binds. Moreover, the debate over the ability of INK4 proteins to associate with or disrupt binary cyclin D-CDK4 complexes in vitro may be largely irrelevant since there is no evidence that such binary complexes ever exist in vivo.
| |
ACKNOWLEDGMENTS |
|---|
We are indebted to Jim Koh, Emma Lees, and Yoichi Taya for providing monoclonal antibodies and to David Parry and Emma Lees for communicating results prior to publication. We also thank Harmut Land and Nic Jones for helpful discussions and comments on the manuscript.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Imperial Cancer Research Fund Laboratories, P.O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Phone: (44) 171 269 3049. Fax: (44) 171 269 3479. E-mail: peters{at}icrf.icnet.uk.
Present address: Department of Radiation Oncology, Emory University
School of Medicine, Atlanta, GA 30335.
Present address: Paterson Institute for Cancer Research,
Manchester M20 9BX, United Kingdom.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
| 1. |
Adachi, M.,
M. F. Roussel,
K. Havenith, and C. J. Sherr.
1997.
Features of macrophage differentiation induced by p19INK4d, a specific inhibitor of cyclin D-dependent kinases.
Blood
90:126-137 |
| 2. |
Alcorta, D. A.,
Y. Xiong,
D. Phelps,
G. Hannon,
D. Beach, and J. C. Barrett.
1996.
Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence.
Proc. Natl. Acad. Sci. USA
93:13742-13747 |
| 3. | Bartkova, J., M. Zemanova, and J. Bartek. 1996. Abundance and subcellular localisation of cyclin D3 in human tumours. Int. J. Cancer 65:323-327[Medline]. |
| 4. | Bates, S., L. Bonetta, D. MacAllan, D. Parry, A. Holder, C. Dickson, and G. Peters. 1994. CDK6 (PLSTIRE) and CDK4 (PSK-J3) are a distinct subset of the cyclin-dependent kinases that associate with cyclin D1. Oncogene 9:71-79[Medline]. |
| 5. |
Blain, S. W.,
E. Montalvo, and J. Massagué.
1997.
Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27KIP1 with cyclin A-Cdk2 and cyclin D2-Cdk4.
J. Biol. Chem.
272:25863-25872 |
| 6. | Byeon, I.-J. L., J. Li, K. Ericson, T. L. Selby, A. Tevelev, H.-J. Kim, P. O'Maille, and M.-D. Tsai. 1998. Tumor suppressor p16INK4A: determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4. Mol. Cell 1:421-431[Medline]. |
| 7. | Chan, F. K. M., J. Zhang, L. Cheng, D. N. Shapiro, and A. Winoto. 1995. Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4. Mol. Cell. Biol. 15:2682-2688[Abstract]. |
| 8. | Chen, J., P. Saha, S. Kornbluth, B. D. Dynlacht, and A. Dutta. 1996. Cyclin-binding motifs are essential for the function of p21CIP1. Mol. Cell. Biol. 16:4673-4682[Abstract]. |
| 9. |
Dai, K.,
R. Kobayashi, and D. Beach.
1996.
Physical interaction of mammalian CDC37 with CDK4.
J. Biol. Chem.
271:22030-22034 |
| 10. |
Della Ragione, F.,
G. L. Russo,
A. Oliva,
C. Mercurio,
S. Mastropietro,
V. Della Pietra, and V. Zappia.
1996.
Biochemical characterization of p16INK4- and p18-containing complexes in human cell lines.
J. Biol. Chem.
271:15942-15949 |
| 11. |
Diller, L.,
J. Kassel,
C. E. Nelson,
M. A. Gryka,
G. Litwak,
M. Gebhardt,
B. Bressac,
M. Ozturk,
S. J. Baker,
B. Vogelstein, and S. H. Friend.
1990.
p53 functions as a cell cycle control point in osteosarcoma.
Mol. Cell. Biol.
10:5772-5781 |
| 12. |
Dimri, G. P.,
X. Lee,
G. Basile,
M. Acosta,
G. Scott,
C. Roskelley,
E. E. Medrano,
M. Linskens,
I. Rubelj,
O. Pereira-Smith,
M. Peacocke, and J. Campisi.
1995.
A novel biomarker identifies senescent human cells in culture and in aging skin in vivo.
Proc. Natl. Acad. Sci. USA
92:9363-9367 |
| 13. |
Dulic, V.,
L. F. Drullinger,
E. Lees,
S. I. Reed, and G. H. Stein.
1993.
Altered regulation of G1 cyclins in senescent human diploid fibroblasts: accumulation of inactive cyclin E-Cdk2 and cyclin D1-Cdk2 complexes.
Proc. Natl. Acad. Sci. USA
90:11034-11038 |
| 14. | Elledge, S. J., J. Winston, and J. W. Harper. 1996. A question of balance: the role of cyclin-kinase inhibitors in development and tumorigenesis. Trends Cell Biol. 6:388-392. [Medline] |
| 15. | Ewen, M. E., H. K. Sluss, C. J. Sherr, H. Matsushime, J. Kato, and D. M. Livingston. 1993. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell 73:487-497[Medline]. |
| 16. | Fotedar, R., P. Fitzgerald, T. Rousselle, D. Cannella, M. Dorée, H. Messier, and A. Fotedar. 1996. p21 contains independent binding sites for cyclin and cdk2: both sites are required to inhibit cdk2 kinase activity. Oncogene 12:2155-2164[Medline]. |
| 17. | Goubin, F., and B. Ducommun. 1995. Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor. Oncogene 10:2281-2287[Medline]. |
| 18. |
Guan, K.-L.,
C. W. Jenkins,
Y. Li,
M. A. Nichols,
X. Wu,
C. L. O'Keefe,
A. G. Matera, and Y. Xiong.
1994.
Growth suppression by p18, a p16INK4/MTS1 and p14INK4/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function.
Genes Dev.
8:2939-2952 |
| 19. | Guan, K.-L., C. W. Jenkins, Y. Li, C. L. O'Keefe, S. Noh, X. Wu, M. Zariwala, A. G. Matera, and Y. Xiong. 1996. Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK4 and CDK6. Mol. Biol. Cell 7:57-70[Abstract]. |
| 20. | Hall, M., S. Bates, and G. Peters. 1995. Evidence for different modes of action of cyclin-dependent kinase inhibitors: p15 and p16 bind to kinases, p21 and p27 bind to cyclins. Oncogene 11:1581-1588[Medline]. |
| 21. |
Hannon, G. J., and D. Beach.
1994.
p15INK4B is a potential effector of TGF--induced cell cycle arrest.
Nature
371:257-261[Medline].
|
| 22. | Hara, E., R. Smith, D. Parry, H. Tahara, S. Stone, and G. Peters. 1996. Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Mol. Cell. Biol. 16:859-867[Abstract]. |
| 23. | Harlow, E., and D. Lane. 1988. Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 24. | Harper, J. W., G. R. Adami, N. Wei, K. Keyomarsi, and S. J. Elledge. 1993. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805-816[Medline]. |
| 25. | Harper, J. W., S. J. Elledge, K. Keyomarsi, B. Dynlacht, L.-H. Tsai, P. Zhang, S. Dobrowolski, C. Bai, L. Connell-Crowley, E. Swindell, M. P. Fox, and N. Wei. 1995. Inhibition of cyclin-dependent kinases by p21. Mol. Biol. Cell 6:387-400[Abstract]. |
| 26. |
Hatakeyama, M.,
J. A. Brill,
G. R. Fink, and R. A. Weinberg.
1994.
Collaboration of G1 cyclins in the functional inactivation of the retinoblastoma protein.
Genes Dev.
8:1759-1771 |
| 27. | Higashi, H., I. Suzuki-Takahashi, S. Saitoh, K. Segawa, Y. Taya, A. Okuyama, S. Nishimura, and M. Kitagawa. 1996. Cyclin-dependent kinase-2 (Cdk2) forms an inactive complex with cyclin D1 since Cdk2 associated with cyclin D1 is not phosphorylated by Cdk7-cyclin H. Eur. J. Biochem. 237:460-467[Medline]. |
| 28. | Hirai, H., M. F. Roussel, J.-Y. Kato, R. A. Ashmun, and C. J. Sherr. 1995. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. Mol. Cell. Biol. 15:2672-2681[Abstract]. |
| 29. |
Jen, J.,
J. W. Harper,
S. H. Bigner,
D. D. Bigner,
N. Papadopoulos,
S. Markowitz,
J. K. V. Willson,
K. W. Kinzler, and B. Vogelstein.
1994.
Deletion of p16 and p15 genes in brain tumors.
Cancer Res.
54:6353-6358 |
| 30. |
Jiang, H.,
H. S. Chou, and L. Zhu.
1998.
Requirement of cyclin E-Cdk2 inhibition in p16INK4a-mediated growth suppression.
Mol. Cell. Biol.
18:5284-5290 |
| 31. | Kitagawa, M., H. Higashi, H.-K. Jung, I. Suzuki-Takahashi, M. Ikeda, K. Tamai, J. Kato, K. Segawa, E. Yoshida, S. Nishimura, and Y. Taya. 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[Medline]. |
| 32. | Koh, J., G. H. Enders, B. D. Dynlacht, and E. Harlow. 1995. Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition. Nature 375:506-510[Medline]. |
| 33. |
LaBaer, J.,
M. D. Garrett,
L. F. Stevenson,
J. M. Slingerland,
C. Sandhu,
H. S. Chou,
A. Fattaey, and E. Harlow.
1997.
New functional activities for the p21 family of CDK inhibitors.
Genes Dev.
11:847-862 |
| 34. | Lamphere, L., F. Fiore, X. Xu, L. Brizuela, S. Keezer, C. Sardet, G. F. Draetta, and J. Gyuris. 1997. Interaction between Cdc37 and Cdk4 in human cells. Oncogene 14:1999-2004[Medline]. |
| 35. |
Lee, M.-H.,
I. Reynisdóttir, and J. Massagué.
1995.
Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution.
Genes Dev.
9:639-649 |
| 36. | Lin, J., C. Reichner, X. Wu, and A. J. Levine. 1996. Analysis of wild-type and mutant p21WAF-1 gene activities. Mol. Cell. Biol. 16:1786-1793[Abstract]. |
| 37. | Loughran, O., A. Malliri, D. Owens, P. H. Gallimore, M. A. Stanley, B. Ozanne, M. C. Frame, and E. K. Parkinson. 1996. Association of CDKN2A/p16INK4A with human head and neck keratinocyte replicative senescence: relationship of dysfunction to immortality and neoplasia. Oncogene 13:561-568[Medline]. |
| 38. | Luh, F. Y., S. J. Archer, P. J. Domaille, B. O. Smith, D. Owen, D. H. Brotherton, A. R. C. Raine, X. Xu, L. Brizuela, S. L. Brenner, and E. D. Laue. 1997. Structure of the cyclin-dependent kinase inhibitor p19Ink4d. Nature 389:999-1003[Medline]. |
| 39. | Lukas, J., J. Bartkova, M. Welcker, O. W. Petersen, G. Peters, M. Strauss, and J. Bartek. 1995. Cyclin D2 is a moderately oscillating nucleoprotein required for G1 phase progression in specific cell types. Oncogene 10:2125-2134[Medline]. |
| 40. | Lukas, J., D. Parry, L. Aagaard, D. J. Mann, J. Bartkova, M. Strauss, G. Peters, and J. Bartek. 1995. Retinoblastoma-protein-dependent cell cycle inhibition by the tumour suppressor p16. Nature 375:503-506[Medline]. |
| 41. |
Lundberg, A. S., and R. A. Weinberg.
1998.
Functional inactivation of the retinobalstoma protein requires sequential modification by at least two distinct cyclin-cdk complexes.
Mol. Cell. Biol.
18:753-761 |
| 42. | Mahony, D., D. A. Parry, and E. Lees. 1998. Active cdk6 complexes are predominantly nuclear and represent only a minority of the cdk6 in T cells. Oncogene 16:603-611[Medline]. |
| 43. |
Matsuoka, S.,
M. C. Edwards,
C. Bai,
S. Parker,
P. Zhang,
A. Baldini,
J. W. Harper, and S. J. Elledge.
1995.
p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene.
Genes Dev.
9:650-662 |
| 44. |
Matsushime, H.,
D. E. Quelle,
S. A. Shurtleff,
M. Shibuya,
C. J. Sherr, and J.-Y. Kato.
1994.
D-type cyclin-dependent kinase activity in mammalian cells.
Mol. Cell. Biol.
14:2066-2076 |
| 45. | McConnell, B. B., M. Starborg, S. Brookes, and G. Peters. 1998. Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. Curr. Biol. 8:351-354[Medline]. |
| 46. | McDonald, N. Q., and G. Peters. 1998. Ankyrin for clues about the function of p16INK4a. Nat. Struct. Biol. 5:85-88[Medline]. |
| 47. |
Medema, R.,
R. E. Herrera,
F. Lam, and R. A. Weinberg.
1995.
Growth suppression by p16INK4 requires functional retinoblastoma protein.
Proc. Natl. Acad. Sci. USA
92:6289-6293 |
| 48. |
Meyerson, M., and E. Harlow.
1994.
Identification of G1 kinase activity for cdk6, a novel cyclin D partner.
Mol. Cell. Biol.
14:2077-2086 |
| 49. |
Musgrove, E. A.,
A. Swarbrick,
C. S. L. Lee,
A. L. Cornish, and R. L. Sutherland.
1998.
Mechanism of cyclin-dependent kinase inactivation by progestins.
Mol. Cell. Biol.
18:1812-1825 |
| 50. | Nakanishi, M., R. S. Robetorye, G. R. Adami, O. M. Pereira-Smith, and J. R. Smith. 1995. Identification of the active region of the DNA synthesis inhibitory gene p21SdiI/CIP1/WAF1. EMBO J. 14:555-563[Medline]. |
| 51. |
Okamoto, A.,
D. J. Demetrick,
E. A. Spillare,
K. Hagiwara,
S. P. Hussain,
W. P. Bennett,
K. Forrester,
B. Gerwin,
M. Serrano,
D. H. Beach, and C. C. Harris.
1994.
Mutations and altered expression of p16INK4 in human cancer.
Proc. Natl. Acad. Sci. USA
91:11045-11049 |
| 52. | Parry, D., S. Bates, D. J. Mann, and G. Peters. 1995. Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product. EMBO J. 14:503-511[Medline]. |
| 53. | Polyak, K., M.-H. Lee, H. Erdjument-Bromage, A. Koff, J. M. Roberts, P. Tempst, and J. Massagué. 1994. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78:59-66[Medline]. |
| 54. | Poon, R. Y. C., H. Toyoshima, and T. Hunter. 1995. Redistribution of the CDK inhibitor p27 between different cyclin-CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation. Mol. Biol. Cell 6:1197-1213[Abstract]. |
| 55. |
Reynisdóttir, I., and J. Massagué.
1997.
The subcellular locations of p15Ink4b and p27Kip1 coordinate their inhibitory interactions with cdk4 and cdk2.
Genes Dev.
11:492-503 |
| 56. |
Reynisdóttir, I.,
K. Polyak,
A. Iavarone, and J. Massagué.
1995.
Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-.
Genes Dev.
9:1831-1845 |
| 57. |
Reznikoff, C. A.,
T. R. Yeager,
C. A. Belair,
E. Savelieva,
J. A. Puthenveettil, and W. M. Stadler.
1996.
Elevated p16 at senescence and loss of p16 at immortalization in human papillomavirus 16 E6, but not E7, transformed human uroepithelial cells.
Cancer Res.
56:2886-2890 |
| 58. | Ruas, M., and G. Peters. 1998. The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim. Biophys. Acta Rev. Cancer 1378:115-177. |
| 59. | Russo, A. A., P. D. Jeffrey, A. K. Patten, J. Massagué, and N. P. Pavletich. 1996. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382:325-331[Medline]. |
| 60. |
Sandhu, C.,
J. Garbe,
N. Bhattacharya,
J. Daksis,
C.-H. Pan,
P. Yaswen,
J. Koh,
J. M. Slingerland, and M. R. Stampfer.
1997.
Transforming growth factor stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells.
Mol. Cell. Biol.
17:2458-2467[Abstract].
|
| 61. |
Serrano, M.,
E. Gómez-Lahoz,
R. A. DePinho,
D. Beach, and D. Bar-Sagi.
1995.
Inhibition of Ras-induced proliferation and cellular transformation by p16INK4.
Science
267:249-252 |
| 62. | Serrano, M., G. J. Hannon, and D. Beach. 1993. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366:704-707[Medline]. |
| 63. |
Shapiro, G. I.,
J. E. Park,
C. D. Edwards,
L. Mao,
A. Merlo,
D. Sidransky,
M. E. Ewen, and B. J. Rollins.
1995.
Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines.
Cancer Res.
55:6200-6209 |
| 64. |
Sherr, C. J.
1996.
Cancer cell cycles.
Science
274:1672-1677 |
| 65. |
Sherr, C. J., and J. M. Roberts.
1995.
Inhibitors of mammalian G1 cyclin-dependent kinases.
Genes Dev.
9:1149-1163 |
| 66. | Soos, T. J., H. Kiyokawa, J. S. Yan, M. S. Rubin, A. Giordano, A. DeBlasio, S. Bottega, B. Wong, J. Mendelsohn, and A. Koff. 1996. Formation of p27-CDK complexes during the human mitotic cell cycle. Cell Growth Differ. 7:135-146[Abstract]. |
| 67. | Steiner, P., A. Philipp, J. Lukas, D. Godden-Kent, M. Pagano, S. Mittnacht, J. Bartek, and M. Eilers. 1995. Identification of a Myc-dependent step during the formation of active G1 cyclin-cdk complexes. EMBO J. 14:4814-4826[Medline]. |
| 68. |
Stepanova, L.,
X. Leng,
S. B. Parker, and J. W. Harper.
1996.
Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4.
Genes Dev.
10:1491-1502 |
| 69. | Stone, S., P. Dayanath, P. Jiang, J. M. Weaver-Feldhaus, S. V. Tavtigian, L. Cannon-Albright, and A. Kamb. 1995. Genomic structure, expression and mutational analysis of the P15 (MTS2) gene. Oncogene 11:987-991[Medline]. |
| 70. | Stott, F. J., S. Bates, M. C. James, B. B. McConnell, M. Starborg, S. Brookes, I. Palmero, E. Hara, K. H. Vousden, and G. Peters. 1998. The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2. EMBO J. 17:5001-5014[Medline]. |
| 71. | Toyoshima, H., and T. Hunter. 1994. p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21. Cell 78:67-74[Medline]. |
| 72. | Uhrbom, L., M. Nistér, and B. Westermark. 1997. Induction of senescence in human malignant glioma cells by p16INK4A. Oncogene 15:505-514[Medline]. |
| 73. | Venkataramani, R., K. Swaminathan, and R. Marmorstein. 1998. Crystal structure of the CDK4/6 inhibitory protein p18INK4c provides insights into ankyrin-like repeat structure/function and tumor-derived p16INK4 mutations. Nat. Struct. Biol. 5:74-81[Medline]. |
| 74. | Vogt, M., C. Haggblom, J. Yeargin, T. Christiansen-Weber, and M. Haas. 1998. Independent induction of senescence by p16INK4a and p21CIP1 in spontaneously immortalized human fibroblasts. Cell Growth Differ. 9:139-146[Abstract]. |
| 75. | Wong, H., and K. Riabowol. 1996. Differential CDK-inhibitor gene expression in aging human diploid fibroblasts. Exp. Gerentol. 31:311-325[Medline]. |
| 76. | Xiong, Y., G. J. Hannon, H. Zhang, D. Casso, R. Kobayashi, and D. Beach. 1993. p21 is a universal inhibitor of cyclin kinases. Nature 366:701-704[Medline]. |
| 77. | Xiong, Y., H. Zhang, and D. Beach. 1992. D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA. Cell 71:505-514[Medline]. |
| 78. | Zhang, H., Y. Xiong, and D. Beach. 1993. Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes. Mol. Biol. Cell 4:897-906[Abstract]. |
Molecular and Cellular Biology, March 1999, p. 1981-1989, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Fiaschi-Taesch, N., Sicari, B., Ubriani, K., Cozar-Castellano, I., Takane, K. K., Stewart, A. F.
(2009). Mutant Parathyroid Hormone-Related Protein, Devoid of the Nuclear Localization Signal, Markedly Inhibits Arterial Smooth Muscle Cell Cycle and Neointima Formation by Coordinate Up-Regulation of p15Ink4b and p27kip1. Endocrinology
150: 1429-1439
[Abstract] [Full Text] -
Macias, E., Miliani de Marval, P. L., De Siervi, A., Conti, C. J., Senderowicz, A. M., Rodriguez-Puebla, M. L.
(2008). CDK2 Activation in Mouse Epidermis Induces Keratinocyte Proliferation but Does Not Affect Skin Tumor Development. Am. J. Pathol.
173: 526-535
[Abstract] [Full Text] -
Majid, S., Kikuno, N., Nelles, J., Noonan, E., Tanaka, Y., Kawamoto, K., Hirata, H., Li, L. C., Zhao, H., Okino, S. T., Place, R. F., Pookot, D., Dahiya, R.
(2008). Genistein Induces the p21WAF1/CIP1 and p16INK4a Tumor Suppressor Genes in Prostate Cancer Cells by Epigenetic Mechanisms Involving Active Chromatin Modification. Cancer Res.
68: 2736-2744
[Abstract] [Full Text] -
James, M. K., Ray, A., Leznova, D., Blain, S. W.
(2008). Differential Modification of p27Kip1 Controls Its Cyclin D-cdk4 Inhibitory Activity. Mol. Cell. Biol.
28: 498-510
[Abstract] [Full Text] -
Jones, R., Ruas, M., Gregory, F., Moulin, S., Delia, D., Manoukian, S., Rowe, J., Brookes, S., Peters, G.
(2007). A CDKN2A Mutation in Familial Melanoma that Abrogates Binding of p16INK4a to CDK4 but not CDK6. Cancer Res.
67: 9134-9141
[Abstract] [Full Text] -
Ruas, M., Gregory, F., Jones, R., Poolman, R., Starborg, M., Rowe, J., Brookes, S., Peters, G.
(2007). CDK4 and CDK6 Delay Senescence by Kinase-Dependent and p16INK4a-Independent Mechanisms. Mol. Cell. Biol.
27: 4273-4282
[Abstract] [Full Text] -
Al-Mohanna, M. A., Al-Khalaf, H. H., Al-Yousef, N., Aboussekhra, A.
(2007). The p16INK4a tumor suppressor controls p21WAF1 induction in response to ultraviolet light. Nucleic Acids Res
35: 223-233
[Abstract] [Full Text] -
Nadella, K. S., Kirschner, L. S.
(2005). Disruption of Protein Kinase A Regulation Causes Immortalization and Dysregulation of D-Type Cyclins. Cancer Res.
65: 10307-10315
[Abstract] [Full Text] -
Han, J., Tsukada, Y.-i., Hara, E., Kitamura, N., Tanaka, T.
(2005). Hepatocyte Growth Factor Induces Redistribution of p21CIP1 and p27KIP1 through ERK-dependent p16INK4a Up-regulation, Leading to Cell Cycle Arrest at G1 in HepG2 Hepatoma Cells. J. Biol. Chem.
280: 31548-31556
[Abstract] [Full Text] -
White, J., Stead, E., Faast, R., Conn, S., Cartwright, P., Dalton, S.
(2005). Developmental Activation of the Rb-E2F Pathway and Establishment of Cell Cycle-regulated Cyclin-dependent Kinase Activity during Embryonic Stem Cell Differentiation. Mol. Biol. Cell
16: 2018-2027
[Abstract] [Full Text] -
Lieman, J. H., Worley, L. A., Harbour, J. W.
(2005). Loss of Rb-E2F Repression Results in Caspase-8-mediated Apoptosis through Inactivation of Focal Adhesion Kinase. J. Biol. Chem.
280: 10484-10490
[Abstract] [Full Text] -
Merched, A. J., Chan, L.
(2004). Absence of p21Waf1/Cip1/Sdi1 Modulates Macrophage Differentiation and Inflammatory Response and Protects Against Atherosclerosis. Circulation
110: 3830-3841
[Abstract] [Full Text] -
Box, A. H., Demetrick, D. J.
(2004). Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines. Carcinogenesis
25: 2325-2335
[Abstract] [Full Text] -
D'Amico, M., Wu, K., Fu, M., Rao, M., Albanese, C., Russell, R. G., Lian, H., Bregman, D., White, M. A., Pestell, R. G.
(2004). The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame (INK4a/ARF) Locus Encoded Proteins p16INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct cis Elements. Cancer Res.
64: 4122-4130
[Abstract] [Full Text] -
Benzeno, S., Narla, G., Allina, J., Cheng, G. Z., Reeves, H. L., Banck, M. S., Odin, J. A., Diehl, J. A., Germain, D., Friedman, S. L.
(2004). Cyclin-Dependent Kinase Inhibition by the KLF6 Tumor Suppressor Protein through Interaction with Cyclin D1. Cancer Res.
64: 3885-3891
[Abstract] [Full Text] -
LaRue, K. E. A., Khalil, M., Freyer, J. P.
(2004). Microenvironmental Regulation of Proliferation in Multicellular Spheroids Is Mediated through Differential Expression of Cyclin-Dependent Kinase Inhibitors. Cancer Res.
64: 1621-1631
[Abstract] [Full Text] -
Matsuda, Y., Ichida, T., Genda, T., Yamagiwa, S., Aoyagi, Y., Asakura, H.
(2003). Loss of p16 Contributes to p27 Sequestration by Cyclin D1-Cyclin-dependent Kinase 4 Complexes and Poor Prognosis in Hepatocellular Carcinoma. Clin. Cancer Res.
9: 3389-3396
[Abstract] [Full Text] -
Ohtani, N., Brennan, P., Gaubatz, S., Sanij, E., Hertzog, P., Wolvetang, E., Ghysdael, J., Rowe, M., Hara, E.
(2003). Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5. JCB
162: 173-183
[Abstract] [Full Text] -
Gonzalez, T., Seoane, M., Caamano, P., Vinuela, J., Dominguez, F., Zalvide, J.
(2003). Inhibition of Cdk4 Activity Enhances Translation of p27kip1 in Quiescent Rb-negative Cells. J. Biol. Chem.
278: 12688-12695
[Abstract] [Full Text] -
Lea, N. C., Orr, S. J., Stoeber, K., Williams, G. H., Lam, E. W.-F., Ibrahim, M. A. A., Mufti, G. J., Thomas, N. S. B.
(2003). Commitment Point during G0->G1 That Controls Entry into the Cell Cycle. Mol. Cell. Biol.
23: 2351-2361
[Abstract] [Full Text] -
Diehl, J. A., Yang, W., Rimerman, R. A., Xiao, H., Emili, A.
(2003). Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase. Mol. Cell. Biol.
23: 1764-1774
[Abstract] [Full Text] -
Obaya, A. J., Kotenko, I., Cole, M. D., Sedivy, J. M.
(2002). The Proto-oncogene c-myc Acts through the Cyclin-dependent Kinase (Cdk) Inhibitor p27Kip1 to Facilitate the Activation of Cdk4/6 and Early G1 Phase Progression. J. Biol. Chem.
277: 31263-31269
[Abstract] [Full Text] -
Rodriguez-Puebla, M. L., Miliani de Marval, P. L., LaCava, M., Moons, D. S., Kiyokawa, H., Conti, C. J.
(2002). cdk4 Deficiency Inhibits Skin Tumor Development but Does Not Affect Normal Keratinocyte Proliferation. Am. J. Pathol.
161: 405-411
[Abstract] [Full Text] -
Gartel, A. L., Tyner, A. L.
(2002). The Role of the Cyclin-dependent Kinase Inhibitor p21 in Apoptosis. Molecular Cancer Therapeutics
1: 639-649
[Abstract] [Full Text] -
Lan, Z., Sever-Chroneos, Z., Strobeck, M. W., Park, C.-H., Baskaran, R., Edelmann, W., Leone, G., Knudsen, E. S.
(2002). DNA Damage Invokes Mismatch Repair-dependent Cyclin D1 Attenuation and Retinoblastoma Signaling Pathways to Inhibit CDK2. J. Biol. Chem.
277: 8372-8381
[Abstract] [Full Text] -
Jensen, S. S., Madsen, M. W., Lukas, J., Binderup, L., Bartek, J.
(2001). Inhibitory Effects of 1{alpha},25-Dihydroxyvitamin D3 on the G1-S Phase-Controlling Machinery. Mol. Endocrinol.
15: 1370-1380
[Abstract] [Full Text] -
Sanchez-Beato, M., Saez, A. I., Navas, I. C., Algara, P., Sol Mateo, M., Villuendas, R., Camacho, F., Sanchez-Aguilera, A., Sanchez, E., Piris, M. A.
(2001). Overall Survival in Aggressive B-Cell Lymphomas Is Dependent on the Accumulation of Alterations in p53, p16, and p27. Am. J. Pathol.
159: 205-213
[Abstract] [Full Text] -
Miliani de Marval, P. L., Gimenez-Conti, I. B., LaCava, M., Martinez, L. A., Conti, C. J., Rodriguez-Puebla, M. L.
(2001). Transgenic Expression of Cyclin-Dependent Kinase 4 Results in Epidermal Hyperplasia, Hypertrophy, and Severe Dermal Fibrosis. Am. J. Pathol.
159: 369-379
[Abstract] [Full Text] -
Hu, B., Mitra, J., van den Heuvel, S., Enders, G. H.
(2001). S and G2 Phase Roles for Cdk2 Revealed by Inducible Expression of a Dominant-Negative Mutant in Human Cells. Mol. Cell. Biol.
21: 2755-2766
[Abstract] [Full Text] -
Foster, J. S., Henley, D. C., Bukovsky, A., Seth, P., Wimalasena, J.
(2001). Multifaceted Regulation of Cell Cycle Progression by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of Cyclin D1-Cdk4 Function. Mol. Cell. Biol.
21: 794-810
[Abstract] [Full Text] -
Jeffrey, P. D., Tong, L., Pavletich, N. P.
(2000). Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors. Genes Dev.
14: 3115-3125
[Abstract] [Full Text] -
Hsieh, F. F., Barnett, L. A., Green, W. F., Freedman, K., Matushansky, I., Skoultchi, A. I., Kelley, L. L.
(2000). Cell cycle exit during terminal erythroid differentiation is associated with accumulation of p27Kip1 and inactivation of cdk2 kinase. Blood
96: 2746-2754
[Abstract] [Full Text] -
Grimison, B., Langan, T. A., Sclafani, R. A.
(2000). p16Ink4a Tumor Suppressor Function in Lung Cancer Cells Involves Cyclin-dependent Kinase 2 Inhibition by Cip/Kip Protein Redistribution. Cell Growth Differ.
11: 507-515
[Abstract] [Full Text] -
van Dyk, L. F., Virgin, H. W. IV, Speck, S. H.
(2000). The Murine Gammaherpesvirus 68 v-Cyclin Is a Critical Regulator of Reactivation from Latency. J. Virol.
74: 7451-7461
[Abstract] [Full Text] -
Stepanova, L., Finegold, M., DeMayo, F., Schmidt, E. V., Harper, J. W.
(2000). The Oncoprotein Kinase Chaperone CDC37 Functions as an Oncogene in Mice and Collaborates with Both c-myc and Cyclin D1 in Transformation of Multiple Tissues. Mol. Cell. Biol.
20: 4462-4473
[Abstract] [Full Text] -
Santoni-Rugiu, E., Falck, J., Mailand, N., Bartek, J., Lukas, J.
(2000). Involvement of Myc Activity in a G1/S-Promoting Mechanism Parallel to the pRb/E2F Pathway. Mol. Cell. Biol.
20: 3497-3509
[Abstract] [Full Text] -
O'Farrell, A.-M., Parry, D. A., Zindy, F., Roussel, M. F., Lees, E., Moore, K. W., Mui, A. L.-F.
(2000). Stat3-Dependent Induction of p19INK4D by IL-10 Contributes to Inhibition of Macrophage Proliferation. J. Immunol.
164: 4607-4615
[Abstract] [Full Text] -
Swarbrick, A., Lee, C. S. L., Sutherland, R. L., Musgrove, E. A.
(2000). Cooperation of p27Kip1 and p18INK4c in Progestin-Mediated Cell Cycle Arrest in T-47D Breast Cancer Cells. Mol. Cell. Biol.
20: 2581-2591
[Abstract] [Full Text] -
Zindy, F., van Deursen, J., Grosveld, G., Sherr, C. J., Roussel, M. F.
(2000). INK4d-Deficient Mice Are Fertile Despite Testicular Atrophy. Mol. Cell. Biol.
20: 372-378
[Abstract] [Full Text] -
Grossel, M. J., Baker, G. L., Hinds, P. W.
(1999). cdk6 Can Shorten G1 Phase Dependent upon the N-terminal INK4 Interaction Domain. J. Biol. Chem.
274: 29960-29967
[Abstract] [Full Text] -
Warner, B. J., Blain, S. W., Seoane, J., Massague, J.
(1999). Myc Downregulation by Transforming Growth Factor beta Required for Activation of the p15Ink4b G1 Arrest Pathway. Mol. Cell. Biol.
19: 5913-5922
[Abstract] [Full Text] -
Sherr, C. J., Roberts, J. M.
(1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev.
13: 1501-1512
[Full Text] -
Mitra, J., Dai, C. Y., Somasundaram, K., El-Deiry, W. S., Satyamoorthy, K., Herlyn, M., Enders, G. H.
(1999). Induction of p21WAF1/CIP1 and Inhibition of Cdk2 Mediated by the Tumor Suppressor p16INK4a. Mol. Cell. Biol.
19: 3916-3928
[Abstract] [Full Text] -
Prall, O. W. J., Carroll, J. S., Sutherland, R. L.
(2001). A Low Abundance Pool of Nascent p21WAF1/Cip1 Is Targeted by Estrogen to Activate Cyclin E{middle dot}Cdk2. J. Biol. Chem.
276: 45433-45442
[Abstract] [Full Text] -
Matushansky, I., Radparvar, F., Skoultchi, A. I.
(2000). Reprogramming leukemic cells to terminal differentiation by inhibiting specific cyclin-dependent kinases in G1. Proc. Natl. Acad. Sci. USA
97: 14317-14322
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»