©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The C-terminal Region of p21 Is Involved in Proliferating Cell Nuclear Antigen Binding but Does Not Appear to Be Required for Growth Inhibition (*)

(Received for publication, May 10, 1995)

Makoto Nakanishi (§) , Ryan S. Robetorye , Olivia M. Pereira-Smith , James R. Smith (¶)

From the Roy M. and Phyllis Gough Huffington Center on Aging, Division of Molecular Virology, and Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030-3498

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cyclin-dependent kinase (Cdk) inhibitor p21 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 p21that 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.


INTRODUCTION

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)()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.

As studies continue with p21, 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.

We have recently identified the Cdk2 binding region of p21and 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.


MATERIALS AND METHODS

Construction of Deletion Mutants of p21

The deletion mutants of p21 were generated by standard PCR techniques using as a template either p21HA cloned into pBluescript (Stratagene) or pCMVp21HA, 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) .

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): 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.

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.

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) . pCMVgal 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 -Galactosidase Activity

cDNAs encoding the deletion mutants (5 µg) were co-transfected with pCMV-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.


RESULTS AND DISCUSSION

In Vitro Binding of p21 to PCNA

To confirm that specific binding occurred between p21 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).


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 [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.


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 [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.




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-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.




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.



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 Proteins That Contain Either Small Deletions or Point Mutations in the Binding Region(s) for Cdk2 and/or PCNA

We constructed mutants that contained deletions in the Cdk2 binding region (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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AG11066, AG07123, and AG05333 and National Institutes of Health Training Grant T32AG00183 (to R. S. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi-ken 329-04, Japan.

To whom correspondence should be addressed: Division of Molecular Virology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498. Tel.: 713-798-4453; Fax: 713-798-4161.

The abbreviations used are: Cdk, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; GST, glutathione S-transferase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis.

G. P. Dimri, M. Nakanishi, J. R. Smith, and J. Campisi, submitted for publication.


ACKNOWLEDGEMENTS

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.


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