(Received for publication, May 10, 1995)
From the
The cyclin-dependent kinase (Cdk) inhibitor
p21
It is becoming increasingly clear that growth control involves
the expression of positive and negative regulatory genes. In studies of
cellular senescence, we and others had accumulated evidence for the
expression of an inhibitor of DNA synthesis in terminally non-dividing
senescent cells(1) . We cloned three such inhibitory cDNAs from
a senescent cell library and further investigated SDI1 because
it was up-regulated at senescence(2, 3) . The same
gene was also cloned as p21(4) , CIP1, a
cyclin-dependent kinase (Cdk)
As studies continue with
p21
We have recently identified the Cdk2 binding
region of p21
The following primer
sets were used to delete the indicated six amino acids and to create a
unique diagnostic SmaI restriction site in the PCR product by
adding the nucleotide sequence CCCCGGGGC in the deleted region (this
resulted in a substitution of the amino acids Pro-Arg-Gly in place of
the six naturally occurring amino acids):
The following primer sets were
used to introduce point mutations, substituting alanine for the
naturally occurring amino acid: K141A, CAGGGTCGAGCACGGCGGCAGACC and
GGTCTGCCGCCGTGCTCGACCCTG; R142A, CAGGGTCGAAAAGCGCGGCAGACC and
GGTCTGCCGCGCTTTTCGACCCTG; R143A, GGTCGAAAACGGGCGCAGACCAGC and
ATGCTGGTCTGCGCCCGTTTTCGA; Q144A, AAAACGGCGGGCGACCAGCATGAC and
GTCATGCTGGTCGCCCGCCGTTTTCGA; T145A, AAACGGCGGCAGGCCAGCATGACA and
ATCTGTCATGCTGGCCTGCCGCCG; S146A, CGGCGGCAGACCGCCATGACAGAT and
ATCTGTCATGGCGGTCTGCCGCCG; M147A, CGGCAGACCAGCGCGACAGATTTC and
GAAATCTGTCGCGCTGGTCTGCCG; T148A, CAGACCAGCATGGCAGATTTCTAC and
GTAGAAATCTGCCATGCTGGTCTG; D149A, ACCAGCATGACAGCTTTCTACCAC and
GTGGTAGAAAGCTGTCATGCTGGT; F150A, AGCATGACAGATGCCTACCACTCC and
GGAGTGGTAGGCATCTGTCATGCT; Y151A, ATGACAGATTTCGCCCACTCCAAA and
TTTGGAGTGGGCGAAATCTGTCAT; H152A, ACAGATTTCTACGCCTCCAAACGC and
CCGGCGTTTGGAGGCGTAGAAATC; S153A, TTCTACCACGCCAAACGCCGGCTG and
CAGCCGGCGTTTGGCGTGGTAGAA; K154A, TTCTACCACTCCGCACGCCGGCTG and
CAGCCGGCGTGCGGAGTGGTAGAA; R155A, TACCACTCCAAAGCCCGGCTGATC and
GATCAGCCGGGCTTTGGAGTGGTA.
The primers used to obtain the W49G point
mutant have been described previously(13) . The W49G +
M147A double point mutant was constructed by PCR using the primer set
for the M147A mutant, described above, and the W49G cDNA as a template.
All constructs were sequenced to verify that the desired deletions or
mutations had been made, that the HA tag was intact, and to confirm
that no additional mutations had been introduced into the constructs.
Figure 1:
Specific binding of p21 to a GST-PCNA
fusion protein. A, wild-type p21 translated in a rabbit
reticulocyte lysate system and labeled with
[
Figure 2:
Mapping of the PCNA binding region of p21. A, various six amino acid deletion mutants of p21 were
translated in a rabbit reticulocyte lysate system and labeled with
[
Figure 4:
In
vivo function of six amino acid deletion and point mutants that lack
Cdk2 and/or PCNA binding regions. A, MDAH041 cells were
transfected by the method described under ``Materials and
Methods,'' using 1 µg of DNA of each indicated deletion mutant
in 35-mm dishes. DNA synthesis inhibition is reported as the average of
three independent experiments, with the indicated standard deviations. B, Western analysis of cells transfected with the indicated
deletion mutant cDNAs. pCMV mutant p21 (10 µg), pCMV
Figure 3:
Site-directed mutagenesis in the PCNA
binding region of p21. A, all point mutations were introduced
by PCR as described under ``Materials and Methods.'' Each
amino acid, indicated by number, was changed to alanine. The indicated
point mutants of p21 were translated, labeled, and bound to GST-PCNA as
described in Fig. 2. Relative binding activity was determined by
measuring the density of the autoradiogram of translation products and
binding to GST-PCNA, followed by normalization to wild-type binding
(100%). B, sequence comparison of the PCNA binding region of
human (3) and mouse (12) p21. Amino acids written in boldface represent those critical for binding to
PCNA.
We thank Susan F. Venable for excellent technical
assistance and Drs. M. Tainsky for use of the MDAH041 cells and D.
Morgan for purified Cdk2.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
has been
found to be involved in cell senescence, cell cycle arrest, and
differentiation. p21
inhibits the activity of
several Cdks, in contrast to other inhibitors such as p15
and p16
, which act on specific
cyclin-Cdk complexes. Of interest were reports that p21
also bound proliferating cell nuclear antigen (PCNA), an
auxiliary protein for DNA polymerase
, and inhibited DNA
replication but not DNA repair in vitro. To better understand
the function of this interaction in vivo, we first determined
the region of p21
that was needed for PCNA
binding. Analysis of deletion mutants of p21
,
which covered the majority of the protein, revealed that deletion of
either amino acids 142-147 or 149-154 resulted in loss of
ability to bind a glutathione S-transferase-PCNA fusion
protein. Site-directed mutagenesis in this region led to the
identification of the PCNA binding motif
RQXXMTXFYXXXR and demonstrated that
mutation of either amino acid Met-147 or Phe-150 resulted in almost
complete ablation of PCNA binding. Interestingly, when we determined
DNA synthesis inhibitory activity of deletion mutants or point mutants
that were unable to bind Cdk2 and/or PCNA, we found that loss of
binding to PCNA did not affect inhibitory activity, whereas lack of
Cdk2 binding greatly reduced the same. This result suggests that the
primary mechanism for inhibition of DNA synthesis by p21
occurs via inhibition of Cdk activity.
(
)inhibitor(5) , WAF1, a
p53-inducible gene(6) , MDA6, a gene involved in
melanocyte differentiation(7) , and CAP20, an
inhibitor of Cdk2 kinase activity(8) . Thus, this gene appears
to play a key negative regulatory role in many instances that involve
loss of cell proliferation.
, the results suggest that the growth
inhibitory activity of this gene might involve multiple mechanisms.
p21
was originally identified as an inhibitor
of DNA synthesis (2, 3) that appeared to act by
inhibition of cyclin-dependent kinases that were active in the G
and S phases of the cell
cycle(4, 5, 8) . The finding that
p21
could also bind proliferating cell nuclear
antigen (PCNA) and inhibit DNA replication but not DNA repair in
vitro suggested that this could be an alternative path for growth
inhibition by the gene(9, 10, 11) . More
recently, p21
has been found to dissociate
E2F1-p107-Cdk complexes and inhibit E2F1-mediated transcription.
(
)In order to understand the in vivo functions of p21
, the functional domains
corresponding to each mechanism need to be identified and subjected to
a mutational analysis.
and demonstrated that mutants
that lack binding to Cdk2 exhibit a significant loss in DNA synthesis
inhibitory activity(13) . In this study we have demonstrated
that amino acids 141-155, with the motif
RQXXMTXFYXXXR, are involved in
binding to PCNA. Interestingly, loss of binding to PCNA did not affect
the DNA synthesis inhibitory activity of the gene.
Construction of Deletion Mutants of
p21
The deletion mutants of
p21 were generated by standard PCR techniques
using as a template either p21
HA cloned into
pBluescript (Stratagene) or pCMVp21
HA, in which
a hemagglutinin (HA) tag sequence was placed in-frame at the C terminus (13) . The primers used to obtain deletion mutants involving
amino acids 1-16, 1-21, 1-31, 1-41,
17-52, 24-29, 30-35, 42-47, 49-53,
53-58, 58-61, 61-66, 66-71, and 72-77
have been described previously(13) .
17-22,
GGCAGCAAGCCCCGGGGCGGCCCAGTGGACAGCGAG and
CACTGGGCCGCCCCGGGGCTTGCTGCCGCATGGGTT;
36-41,
GACTGTGATCCCCGGGGCATCCAGGAGGCCCGTGAG and
CTCCTGGATGCCCCGGGGATCACAGTCGCGGCTCAG;
79-84,
CTCTACCTTCCCCGGGGCGGCCGGGATGAGTTGGGA and
ATCCCGGCCGCCCCGGGGAAGGTAGAGCTTGGGCAG;
86-91,
CGGCGAGGCCCCCGGGGCGGCAGGCGGCCTGGCACC and
CCGCCTGCCGCCCCGGGGGCCTCGCCGGGGCCCCGT;
93-98,
GGAGGAGGCCCCCGGGGCCCTGCTCTGCTGCAGGGGAC and
CAGAGCAGGGCCCCGGGGGCCTCCTCCCAACTCATC;
100-105,
ACCTCACCTCCCCGGGGCGCAGAGGAAGACCATGTG and
TTCCTCTGCGCCCCGGGGAGGTGAGGTGCCAGGCCG;
107-112,
GGGACAGCACCCCGGGGCCTGTCACTGTCTTGTACC and
CAGTGACAGGCCCCGGGGTGCTGTCCCCTGCAGCAG;
114-119,
GTGGACCTGCCCCGGGGCGTGCCTCGCTCAGGGGAG and
GCGAGGCACGCCCCGGGGCAGGTCCACATGGTCTTCCTCTG;
121-126,
ACCCTTGTGCCCCGGGGCGCTGAAGGGTCCCCAGGT and
CCCTTCAGCGCCCCGGGGCACAAGGGTACAAGACAG;
128-133,
GAGCAGGCTCCCCGGGGCCCTGGAGACTCTCAGGGT and
GTCTCCAGGGCCCCGGGGAGCCTGCTCCCCTGAGCG;
135-140,
GGTGGACCTCCCCGGGGCAAACGGCGGCAGACCAGC and
CCGCCGTTTGCCCCGGGGAGGTCCACCTGGGGACCC;
142-147,
GGTCGAAAACCCCGGGGCACAGATTTCTACCACTCC and
GAAATCTGTGCCCCGGGGTTTTCGACCCTGAGAGTC;
149-154,
AGCATGACACCCCGGGGCCGCCGGCTGATCTTCTCC and
CAGCCGGCGGCCCCGGGGTGTCATGCTGGTCTGCCG;
156-161,
TCCAAACGCCCCCGGGGCAGGAAGCCCTACCCATACG and
GGGCTTCCTGCCCCGGGGGCGTTTGGAGTGGTAGAA.
Construction of the Glutathione S-transferase (GST)-PCNA
Fusion Protein
The human PCNA cDNA was amplified by PCR from
total RNA isolated from CSC303 cells, a human neonatal foreskin
fibroblast cell line (14) using PCR primers
CGTGGATCCCACTCCGCCACCATGTTCGAG and GCTGGGATCCTAGAAGCAGTTCTCAAAGAG. BamHI sites were included in the primers so that after BamHI digestion of the PCR product it could be cloned in-frame
into the BamHI cloning site of the pGEX-2T GST fusion protein
vector (Pharmacia Biotech Inc.). The PCNA cDNA was sequenced to ensure
the accuracy of the PCR amplification procedure.
In Vitro Binding of GST-PCNA and Cdk2 to
p21
The various deletion or
site-directed mutants of p21 were translated in
reticulocyte lysates (Promega) using pBluescript-based plasmids as
transcription templates for T7 RNA polymerase. Labeling of translation
products was performed using 40 µCi of
[
S]methionine (1100 Ci/mM) (ICN) in a
50-µl reaction. 5 µl of each translation reaction was analyzed
by SDS-PAGE, and the remaining 45 µl of the translation product was
used for in vitro binding assays, which contained 500 µl
of binding buffer (50 mM Tris-HCl, pH 7.5, 120 mM
NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 1 mM NaF, 0.1
mM sodium vanadate, 5 µg/ml leupeptin, 5 µg/ml soybean
trypsin inhibitor, 5 µg/ml of aprotinin) and the indicated amount
of GST-PCNA fusion protein or GST protein alone. The mixture was gently
rocked for 1 h at 4 °C and then absorbed by incubation for 1 h with
40 µl of glutathione-conjugated agarose beads (Sigma). The beads
were then washed four times with 0.5 ml of binding buffer prior to
electrophoresis and autoradiography to detect the
S-labeled p21
. Cdk2 binding assays
were performed as described previously(13) . Cdk2 protein
(provided by D. Morgan) for binding assays was purified from a
baculovirus expression system(15) .
Cell Culture, Transfection, and Determination of DNA
Synthesis Inhibitory Activity
MDAH041, immortal human skin
fibroblast cells, were derived from a patient with Li-Fraumeni
Syndrome. These cells do not express an active p53 gene product as a
frameshift mutation causes premature termination in the N-terminal
region of the molecule(16) . Details of cell culture conditions
have been described previously(14) . MDAH041 cells in 35-mm
dishes were co-transfected with 1 µg of pCMV-gal and 1 µg
of the plasmid DNAs described above using calcium phosphate
precipitation(17) . pCMV
gal was used as a marker to detect
transfected cells. Tritiated thymidine (1 µCi/ml) was added to the
culture medium 24 h after transfection, and the cells were incubated
for an additional 24 h. The cells were fixed, stained for
-galactosidase activity, and processed for autoradiography to
determine the percentage of
-galactosidase positive cells that had
synthesized DNA(13) . The percentage inhibition was determined
relative to control cells co-transfected with pCMV vector and
pCMV
-gal.
Western Analysis and Measurement of
cDNAs encoding the deletion mutants (5 µg) were
co-transfected with pCMV--Galactosidase
Activity
gal into MDAH041 cells in 100-mm dishes
using the calcium phosphate precipitation method described above. After
24 h the cells were harvested in 500 µl of reporter lysis buffer
(Promega). In order to normalize the cell lysate for transfection
efficiency, 50 µl of the extract was used for assay of
-galactosidase activity to calculate extract loading volumes
(based on an equivalent amount of
-galactosidase activity) for
SDS-PAGE (16.5% acrylamide). Following transfer to Immobilon membranes
(Millipore), proteins were detected by incubation with the anti-HA
monoclonal antibody 12CA5 (1:1500; BabCo) and enhanced
chemiluminescence (ECL, Amersham Corp.) using Kodak XAR-5 film.
In Vitro Binding of p21
To confirm that specific binding occurred between
p21 to
PCNA
and PCNA, we produced wild-type
p21
protein in an in vitro translation
system. The protein was
S-labeled and incubated with
different amounts of a GST-PCNA fusion protein or GST alone, and the
resulting complexes were analyzed by SDS-PAGE followed by
autoradiography. The wild-type protein bound the GST-PCNA fusion
protein in a concentration-dependent manner (Fig. 1A),
and the specificity of the p21
-PCNA interaction
was confirmed by incubation with GST protein alone. In vitro translated p21
failed to bind GST protein
even when as much as 2 µg of the protein was added (Fig. 1A).
S]methionine during translation was incubated
with the indicated amount of GST or GST-PCNA fusion protein in binding
buffer, as described under ``Materials and Methods.'' The
resulting complexes were absorbed with glutathione-agarose beads and
then analyzed by SDS-PAGE. B, in vitro translated
wild-type p21 was incubated with 0.8 µg of GST-PCNA and the
indicated amount of purified Cdk2 produced from a baculovirus
expression system. The resulting complexes were absorbed and analyzed
by the same methods described in A.
Since we had previously found that in
vitro translated p21 bound to purified
Cdk2 in vitro(13) , we wished to determine whether
interaction with Cdk2 affected binding to PCNA. The addition of various
amounts of purified Cdk2 to the binding reaction was found to have no
apparent effect on binding to PCNA (Fig. 1B),
suggesting that p21
could bind to PCNA
independently and also that Cdk and PCNA binding occurred
simultaneously. This hypothesis is supported by the results of Xiong et al.(4) , who found that increasing amounts of
p21
resulted in an increased amount of PCNA in
the cyclin-Cdk-p21
-PCNA complex.
Mapping of the PCNA-binding Region in
p21
In order to identify the region
required for association with PCNA, various deletion mutants of
p21 were constructed by PCR and
S-labeled mutant proteins produced by an in vitro translation system. Translation products were tested for the
ability to bind to GST-PCNA in vitro and then analyzed by
SDS-PAGE followed by autoradiography (Fig. 2, A and B). Only two deletions, of amino acids 142-147 and
149-154, had no ability to bind to PCNA, and deletion of amino
acids 156-161 resulted in weak binding (Fig. 2A,
15% of wild type). The other deletions had almost the same binding
activity as wild type (Fig. 2, A and B). Since
the N-terminal truncated mutants, deleted in amino acids 1-16,
1-21, 1-31, and 1-41, and a large internal deletion
of amino acids 17-52 also bound to PCNA as well as wild type (Fig. 2B), the results indicated that the PCNA binding
region was between amino acids 141 and 155. Deletions in amino acids
49-53, 53-58, 58-61, 61-66, and 66-71,
which fail to bind to Cdk2 (Fig. 4C, (13) , and
data not shown), showed almost the same activity as wild type (Fig. 2A) suggesting that the PCNA binding domain did
not overlap with the Cdk2 binding region. Interestingly, the PCNA
region overlaps with a putative nuclear localization signal in the
C-terminal region (3, 4, 5, 6, 8) . However,
we have found that this region is not necessary for translocation to
the nucleus or DNA synthesis inhibitory activity(13) .
Nonetheless, this region is highly conserved between the human and
mouse sequence (Fig. 3B)(12) , indicating a
potential role in the activity of the p21
gene.
S]methionine, and the translated products were
confirmed by SDS-PAGE (upperpanel). In vitro translated mutant proteins were incubated with 2 µg of
GST-PCNA fusion protein as described under ``Materials and
Methods.'' The resulting complexes were absorbed with
glutathione-agarose beads and then analyzed by SDS-PAGE (lowerpanel). B, p21 mutants with large amino-terminal
deletions were translated in vitro (upperpanel) and analyzed for binding to PCNA (lowerpanel) as described in A.
-gal (5
µg), and pBluescript (10 µg) as carrier DNA were transfected
into 100-mm dishes at 50-70% confluency. Extracts were loaded on
the basis of equal amounts of
-galactosidase activity to normalize
transfection efficiency. Proteins were detected with anti-HA tag
antibodies as described under ``Materials and Methods.'' C, analysis of the p21 mutants described in A for
binding to Cdk2. Each of the various mutants was translated in
vitro (upperpanel) and analyzed by SDS-PAGE for
binding to Cdk2 (lowerpanel) as described under
``Materials and Methods.'' D, MDAH041 cells were
transfected with 1 µg of DNA of each indicated point mutant as
described in A.
Site-directed Mutagenesis in the Region of Amino Acids
141-155 of p21
Since the native
structure of the p21protein could potentially
be adversely affected by the creation of small deletions, we then
performed site-directed mutagenesis in the region of amino acids
141-155. The amount of product translated in vitro was
determined for each point mutant, as well as the ability of the in
vitro translated proteins to bind PCNA (Fig. 3A).
Since the amount of protein produced by each mutant varied, the binding
capacity was normalized to the amount of product synthesized. The
results demonstrate that mutation M147A and F150A resulted in an almost
complete loss of binding to PCNA. Significant loss (>50%) of binding
was also caused by mutation of residues 144, 148, 151, and 155.
Interestingly, mutation of residues 141, 142, 146, 152, and 153
resulted in almost no loss of binding ability. Thus, the sequence of
the PCNA binding region of p21
encompasses
amino acids 141-155, with the specific amino acids involved in
binding denoting the motif
RQXXMTXFYXXXR (Fig. 3B). This sequence has no significant sequence
similarity to other proteins in the data base, and in particular to
cyclin D1 and GADD 45, both of which have been shown to bind to
PCNA(18, 19) . This suggests that the binding site,
and perhaps the mechanism of binding to p21
, is
different from that of other proteins, and the fact that both amino
acids 147 (methionine) and 150 (phenylalanine) are hydrophobic suggests
that the interaction is hydrophobic. This is supported by the fact that
in the mouse cDNA methionine 147 has been substituted by another
hydrophobic amino acid, leucine (Fig. 3B).
In Vivo DNA Synthesis Inhibitory Activity of Mutant
p21
We
constructed mutants that contained deletions in the Cdk2 binding region
( Proteins That Contain Either Small Deletions or
Point Mutations in the Binding Region(s) for Cdk2 and/or PCNA
49-53 and
17-52), the PCNA binding region
(
142-147), and both binding regions (
49-53 and
142-147) in a mammalian expression vector driven by the
cytomegalovirus promoter (pCMV vector). These were transfected into
MDAH041 cells, which do not express detectable levels of endogenous
p21
(16) , and therefore provided a
sensitive assay for small changes in DNA synthesis inhibitory activity
that might be expected when these mutants were overexpressed.
Transfection of the full-length coding region resulted in a
95%
decrease in the percentage of cells incorporating tritiated thymidine (Fig. 4A). Deletion mutant
17-52, which
failed to bind to Cdk2 (Fig. 4C) but bound to PCNA (Fig. 2B), did not exhibit any significant DNA
synthesis inhibitory activity (Fig. 4A). On the other
hand, deletion of amino acids 49-53 resulted in decreased Cdk2
binding (Fig. 4C) and decreased DNA synthesis
inhibitory activity to about 50% of wild type (Fig. 4A), but binding of PCNA was similar to that of
wild-type p21
(Fig. 2A).
Deletion of amino acids 142-147, which contained amino acids
important for PCNA binding (Fig. 2A), did not result in
any significant loss of DNA synthesis inhibitory activity (Fig. 4A) or Cdk2 binding (Fig. 4C). In
order to determine whether inhibition of DNA replication required
binding to PCNA, we constructed a double deletion mutant deficient in
amino acids 49-53, required for Cdk2 binding (Fig. 4C), and amino acids 142-147, required for
PCNA binding (Fig. 2A). Following transfection into
MDAH041 cells, this double deletion mutant had almost the same DNA
synthesis inhibitory activity as that of deletion 49-53 alone (Fig. 4A), suggesting that a mechanism other than that
involving PCNA binding was responsible for the inhibitory activity. One
possible explanation for this result is that the presence of the
deletions, although relatively small, might result in a gross
alteration in protein folding that prevented p21
from exhibiting its full inhibitory capacity. In order to
rule out this possibility, we analyzed point mutants in amino acids
that had previously been determined to be important for Cdk2 and PCNA
binding. Transfection of the W49G mutant, which has reduced Cdk2
binding (data not shown), resulted in very low DNA synthesis inhibitory
activity (Fig. 4D), whereas transfection of the M147A
mutant, which does not bind PCNA (Fig. 3A), resulted in
inhibition of DNA synthesis equivalent to that observed for wild-type
p21
(Fig. 4D). A double mutant,
containing both the W49G and M147A mutations, had very low inhibitory
activity, similar to that observed for the W49G mutant (Fig. 4D). These results support the hypothesis that
p21
-mediated inhibition of DNA synthesis is
strongly dependent upon Cdk2 binding and does not appear to require
PCNA binding. Thus, inhibition of DNA replication by binding to PCNA is
probably not the primary mode of DNA synthesis inhibition by
p21
during the normal cell cycle but rather may
be required only in special instances such as DNA damage or progression
to tumorigenicity. The potential multifunctional aspects of this gene
will only become clearer from future studies.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.