The Post-translational Modifications of Proliferating Cell Nuclear Antigen

The diverse function of proliferating cell nuclear antigen (PCNA) is thought to be due, in large part, to post-translational modifications. Here we show by high resolution two-dimensional PAGE analysis that there are three distinct PCNA isoforms that differ in their acetylation status. The moderately acetylated main (M) form was found in all of the subcellular compartments of cycling cells, whereas the highly acetylated acidic form was primarily found in the nucleoplasm, nuclear matrix, and chromatin. Interestingly, the deacetylated basic form was most pronounced in the nucleoplasm of cycling cells. The cells in G0 and the cytoplasm of cycling cells contained primarily the M form only. Because p300 and histone deacetylase (HDAC1) were co-immunoprecipitated with PCNA, they are likely responsible for the acetylation and deacetylation of PCNA, respectively. We also found that deacetylation reduced the ability of PCNA to bind to DNA polymerases β and δ. Taken together, our data support a model where the acidic and M forms participate in DNA replication, whereas the basic form is associated with the termination of DNA replication. The diverse function of proliferating cell nuclear antigen (PCNA) is thought to be due, in large part, to post-translational modifications. Here we show by high resolution two-dimensional PAGE analysis that there are three distinct PCNA isoforms that differ in their acetylation status. The moderately acetylated main (M) form was found in all of the subcellular compartments of cycling cells, whereas the highly acetylated acidic form was primarily found in the nucleoplasm, nuclear matrix, and chromatin. Interestingly, the deacetylated basic form was most pronounced in the nucleoplasm of cycling cells. The cells in G0 and the cytoplasm of cycling cells contained primarily the M form only. Because p300 and histone deacetylase (HDAC1) were co-immunoprecipitated with PCNA, they are likely responsible for the acetylation and deacetylation of PCNA, respectively. We also found that deacetylation reduced the ability of PCNA to bind to DNA polymerases β and δ. Taken together, our data support a model where the acidic and M forms participate in DNA replication, whereas the basic form is associated with the termination of DNA replication. PCNA 1The abbreviations used are: PCNA, proliferating cell nuclear antigen; CHO, Chinese hamster ovary cell; HDAC, histone deacetylase; TSA, trichostatin A; MEM, minimal essential medium; NEF, nuclear extract fraction (i.e. soluble nuclear fraction); CMF, chromatin fraction; NMF, nuclear matrix fraction; CF, cytosol fraction; IEF, isoelectrofocusing; M, main; A, acidic; B, basic; PVDF, polyvinylidene difluoride; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid; DTT, dithiothreitol. 1The abbreviations used are: PCNA, proliferating cell nuclear antigen; CHO, Chinese hamster ovary cell; HDAC, histone deacetylase; TSA, trichostatin A; MEM, minimal essential medium; NEF, nuclear extract fraction (i.e. soluble nuclear fraction); CMF, chromatin fraction; NMF, nuclear matrix fraction; CF, cytosol fraction; IEF, isoelectrofocusing; M, main; A, acidic; B, basic; PVDF, polyvinylidene difluoride; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid; DTT, dithiothreitol. participates in a broad spectrum of biological activities, including DNA replication, repair, cell cycle control, apoptosis, and chromatin remodeling (1Tsurimoto T. Front. Biosci. 1999; 4: D849-D858Crossref PubMed Google Scholar, 2Stucki M. Stagljar I. Jonsson Z.O. Hubscher U. Prog. Nucleic Acids Res. Mol. Biol. 2001; 65: 261-298Crossref PubMed Google Scholar, 3Ellison V. Stillman B. Cell. 2001; 106: 655-660Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Several reports (4Bravo R. Macdonald-Bravo H. J. Cell Biol. 1987; 105: 1549-1554Crossref PubMed Scopus (832) Google Scholar, 5McCormick D. Hall P.A. Histopathology. 1992; 21: 591-594Crossref PubMed Scopus (245) Google Scholar, 6Tanno M. Taguchi T. Exp. Mol. Pathol. 1999; 67: 192-200Crossref PubMed Scopus (15) Google Scholar, 7Brand S.R. Bernstein R.M. Mathews M.B. J. Immunol. 1994; 53: 3070-3078Google Scholar) previously suggested the existence of different PCNA species, which is thought to be relevant to the diverse functions of PCNA. Furthermore, we have shown recently by high resolution two-dimensional PAGE and immunoblot analyses that both hamster and human cells contain three different PCNA isoforms, the acidic (A), the main (M), and the basic (B) forms (8Naryzhny S.N. Lee H. Proteomics. 2003; 3: 930-936Crossref PubMed Scopus (25) Google Scholar). The different PCNA isoforms could be the result of post-translational modifications, especially phosphorylation status (9Kelman Z. Oncogene. 1997; 14: 629-640Crossref PubMed Scopus (712) Google Scholar, 10Jonsson Z.O. Hubscher U. BioEssays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar, 11Prosperi E. Prog. Cell Cycle Res. 1997; 3: 193-210Crossref PubMed Scopus (115) Google Scholar, 12Loor G. Zhang S.J. Zhang P. Toomey N.L. Lee M.Y. Nucleic Acids Res. 1997; 25: 5036-5041Crossref Scopus (76) Google Scholar). Consistent with this hypothesis, Prosperi et al. (13Prosperi E. Scovassi A.I. Stivala L.A. Bianchi L. Exp. Cell Res. 1994; 215: 257-262Crossref PubMed Scopus (67) Google Scholar) presented data that support the phosphorylation of PCNA. In contrast, however, Bravo and Celis (14Bravo R. Celis J. FEBS Lett. 1985; 182: 435-440Crossref PubMed Scopus (34) Google Scholar) did not find any evidence of PCNA phosphorylation. We wanted to address this issue by carrying out several different experimental approaches, including in vivo labeling, immunoprecipitation, and immunoblot analysis in combination with high resolution two-dimensional PAGE. Here we show that mammalian PCNA is regulated not by phosphorylation but by acetylation. We have also shown that three distinct PCNA isoforms differ in their acetylation status. Our data further show that acetylation is associated with subcellular localization of PCNA. Moreover, deacetylated PCNA by HDAC1 has lower affinity to DNA polymerases β and δ than that of PCNA treated with trichostatin A (TSA, an HDAC inhibitor). Most important, the PCNA-DNA polymerase complex treated with HDAC1 showed lower polymerization activity than a TSA-treated control. Taken together, our data are consistent with the idea that acetylated PCNA is involved in the DNA replication process, whereas deacetylation of PCNA is associated with the termination of DNA replication. Reagents—All the reagents used are from Sigma unless stated otherwise. The suppliers of other reagents are as follows: protease inhibitor mixture, Roche Diagnostics; bovine histone deacetylase, Calbiochem; IPG gel strips, IPG buffers, DryStrip cover fluid, polyvinylidene difluoride (PVDF) membrane, solution for the ECL reaction, and poly(dA)-(dT)12–18, Amersham Biosciences; dialyzed fetal bovine serum, Invitrogen; anti-PCNA monoclonal antibody PC10, NeoMarkers (Fremont, CA); anti-PCNA-AC monoclonal antibodies (agarose-conjugated), polyclonal anti-HDAC1, anti-DNA polymerases β and δ antibodies, peroxidase-conjugated secondary anti-goat antibodies, protein A/G-agarose beads, and normal mouse IgG, Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-acetyl protein antibody, Novus Biologicals (Littleton, CO); peroxidase-conjugated secondary anti-rabbit antibodies, Pierce; [32P]orthophosphate, [3H]sodium acetate, and [32P]dTTP, PerkinElmer Life Sciences. Cell-cycle Synchronization—The arrests of cell cycle in G0 and at the G1/S boundary were achieved by isoleucine deprivation and by the combination of isoleucine starvation and mimosine treatment, respectively (15Naryzhny S.N. Lee H. Electrophoresis. 2001; 22: 1764-1775Crossref PubMed Scopus (40) Google Scholar, 16Lee H. Larner J.M. Hamlin J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 526-531Crossref PubMed Scopus (40) Google Scholar). In Vivo Labeling—For phosphoprotein labeling, CHO cells at 60–70% confluence were maintained for 4 h in phosphate-omitted MEM (Invitrogen, catalog number 21097-035) containing 10% dialyzed fetal bovine serum (Invitrogen). Subsequently, the cells were further incubated in fresh MEM containing [32P]orthophosphate (0.4 mCi/ml) for 9 h at 37 °C. Cells were then rinsed twice with cold PBS, collected using a plastic cell lifter in PBS containing protease inhibitor mixture and phosphatase inhibitors (20 mm NaF and 1 mm Na3VO4), and then harvested by centrifugation at 1,000 × g for 5 min. For acetylation experiments, cells were incubated for 6 h in MEM containing [3H]sodium acetate (0.5 mCi/ml). Subcellular Fractionation—Protein isolation and subcellular fractionation were as described previously (15Naryzhny S.N. Lee H. Electrophoresis. 2001; 22: 1764-1775Crossref PubMed Scopus (40) Google Scholar, 17Spector D.L. Goldman R.D. Leinwand L.A. The Nuclear Matrix: Preparation for Microscopy and Biochemical. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 44.1-44.4Google Scholar, 18Ramsby M. Makowski G. Link A.J. 2-D Proteome Analysis Protocol. Humana Press Inc., Totowa, NJ1999: 53-66Google Scholar). One notable modification was the use of digitonin in the preparation of cytosol fraction. Briefly, cells in monolayer were washed once with ice-cold PBS and treated for 10 min on ice with 1 ml of digitonin extraction buffer (10 mm PIPES, pH 6.8, 0.015% digitonin, 300 mm sucrose, 100 mm NaCl, 3 mm MgCl2, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture) per 10-cm flask. The cells were then scraped using a plastic cell lifter, and nuclei were pelleted by centrifugation at 2,000 × g for 5 min. The supernatant was collected as cytosol fraction. All procedures were carried out on ice to minimize protein degradation. All buffers also contained phosphatase inhibitors (20 mm NaF and 1 mm Na3VO4). Two-dimensional Gel Electrophoresis—The procedure was as described previously (8Naryzhny S.N. Lee H. Proteomics. 2003; 3: 930-936Crossref PubMed Scopus (25) Google Scholar, 15Naryzhny S.N. Lee H. Electrophoresis. 2001; 22: 1764-1775Crossref PubMed Scopus (40) Google Scholar). Briefly, samples (up to 2 mg of protein) were typically solubilized in lysis buffer (LB) (9 m urea, 4% CHAPS, 1% DTT, 2% IPG buffer, pH 3–10, protease inhibitors mixture, 0.1 mm MG132 proteosome inhibitor, 0.001% bromphenol blue). Protein separation by isoelectrofocusing (IEF) gel electrophoresis was carried out using a DryStrip kit (Amersham Biosciences) according to the following protocol suggested by the manufacturer's suggestions. Samples in LB were mixed with rehydration solution (8 m urea, 2% CHAPS, 0.3% DTT, 2% IPG buffer, pH range same as in the strip, 0.001% bromphenol blue) in a total volume of 200 μl (7-cm strip), 300 μl (13-cm strip), or 450 μl (18-cm strip). To prepare strips for separation in first direction, IPG gel strips were rehydrated overnight at 10 °C by placing them “upside down” in rehydration solution in the Immobiline DryStrip Reswelling Tray (Amersham Biosciences). 7-cm (pH 4.0–7.0), 13-cm (pH 4.0–7.0), 13-cm (pH 3.0–10.0), or 18-cm (pH 4.0–5.0) strips were used. IEF was conducted at 20 °C using a Multiphor II unit with the DryStrip kit. The total voltage hours were 18,000 and 38,000 for 13- or 18-cm IPG gel strips, respectively. Protein Staining and Gel Drying—Proteins in the gel were “silverstained” as described previously (19Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3736) Google Scholar, 20Rabilloud T. Electrophoresis. 1992; 13: 429-439Crossref PubMed Scopus (247) Google Scholar) with some modifications. Briefly, a polyacrylamide gel was fixed for 15 min in 25% isopropyl alcohol and 10% acetic acid solution, which was repeated at least once more. The gel was rinsed in water for 10 min and then treated with sodium thiosulfate (0.2 g/liter) for 1 min. After rinsing twice in water (20 s for each time), the gel was incubated in silver nitrate (2.0 g/liter) for 30 min. After washing once with water (20 s) and once for 30 s with Developing Solution (sodium carbonate, 30 g/liter, formaldehyde, 1.4 ml of 37% solution/liter, and sodium thiosulfate, 10 mg/liter), protein bands/spots were visualized by further treating the gel with Developing Solution. The reaction was stopped with 10% acetic acid solution. Finally, the gel was dried in the heat oven after it was placed between two sheets of cellophane (Bio-Rad) fastened in two frames as described previously (15Naryzhny S.N. Lee H. Electrophoresis. 2001; 22: 1764-1775Crossref PubMed Scopus (40) Google Scholar). Other Procedures—Immunoprecipitation was carried out according to a protocol supplied by Santa Cruz Biotechnology with some modifications. Briefly, cells on the plate were washed with PBS, scraped with a plastic cell lifter in PBS, and pelleted by centrifugation for 5 min at 1,000 × g. After adding 1–3 ml of ice-cold RIPA buffer (for ∼7 × 107 cells), cells were disrupted by repeated aspiration using a pipette. The cells in RIPA buffer were then incubated on ice for 15 min. Cell debris was removed by centrifugation for 10 min at 10,000 × g at 4 °C. To preclear, the cell lysate was mixed with 1.0 μg of control IgG (i.e. corresponding to the host species primary antibody) and then 20 μl of protein A/G-agarose, followed by centrifugation for 5 min at 3,000 × g at 4 °C. The supernatant (which contains ∼1.0 mg of proteins) was incubated with 1.0–10 μl (0.2–2.0 μg) of primary antibody for 1–2 h at 4 °C, after which 20 μl of protein A/G-agarose was added. After incubation for ≥2 h at 4 °C on a rocker platform, immunoprecipitate was collected by centrifugation at 3,000 × g for 5 min at 4 °C. The pellet was washed 2–4 times with 1.0 ml of PBS or RIPA buffer (for more stringent wash) under the same centrifugation conditions. All the buffers contained protease inhibitor cocktail and phosphatase inhibitors as described above. Immunoblot analysis was as described previously (8Naryzhny S.N. Lee H. Proteomics. 2003; 3: 930-936Crossref PubMed Scopus (25) Google Scholar, 21Mansfield M. Macdonald C.G. Millipore Technical Note TN051. Millipore, Billerica, MA1999Google Scholar). The treatments of HDAC1 and TSA were as recommended by the supplier (Calbiochem). An assay for DNA polymerization activity was carried out according to Podust et al. (22Podust L.M. Podust V.N. Sogo J.M. Hubscher U. Mol. Cell. Biol. 1995; 15: 3072-3081Crossref PubMed Scopus (95) Google Scholar) with some modifications. Briefly, PCNA (complexes) immunoprecipitated by PC10 was eluted by 0.1 m glycine, 0.15 m NaCl, pH 2.7, after which the pH was adjusted to pH 7.0 using 1.0 m Tris (final concentration, 25 mm). In vitro DNA polymerization assay was carried out in 25 μl of sample containing PCNA immunoprecipitate (50 ng of protein), 0.025 units of the poly(dA) template-oligo(dT) primer mixture, 50 mm Tris, pH 6.8, 0.25 mg/ml bovine serum albumin, 1 mm DTT, 6 mm MgCl2, 10 mm 1 mm of at 37 °C. was and DNA was by 1.0 ml of cold acid containing 1% sodium The was collected on and in a PCNA in from Chinese hamster ovary cells were by two-dimensional a membrane, and then using a mixture of and antibodies The same membrane, was then with PC10 anti-PCNA monoclonal antibody PCNA by PC10 in was not by antibodies in that hamster PCNA is not To further the phosphorylation of PCNA, from the cells that with [32P]orthophosphate were by two-dimensional PAGE the of the protein in 1 and our data (8Naryzhny S.N. Lee H. Proteomics. 2003; 3: 930-936Crossref PubMed Scopus (25) Google Scholar, 15Naryzhny S.N. Lee H. Electrophoresis. 2001; 22: 1764-1775Crossref PubMed Scopus (40) Google the of PCNA could be in The same was to an Consistent with the data in PCNA could be in of in vivo that PCNA is not from the cells that with [32P]orthophosphate were by two-dimensional PAGE pH 13-cm gel gel strip). The gel was and dried and to an and the containing PCNA were the PCNA To further our PCNA (complexes) was immunoprecipitated with PC10 anti-PCNA monoclonal The immunoprecipitated were then by two-dimensional followed by silver The same gel was also to an was that the main form of PCNA in was not in there was a phosphoprotein the PCNA in A and the was in this protein was highly in To further this we the PC10 immunoprecipitate by high resolution two-dimensional PAGE pH 18-cm IPG of the gel were a membrane, which was to an The same was then to with PC10 by which all three PCNA isoforms were PCNA was not in with resolution two-dimensional PAGE in the of a phosphoprotein was in phosphoprotein which was not by PC10, was and more acidic than the PCNA A form or the M form phosphoprotein was by PC10 and could be for PCNA following a resolution gel Taken together, we have that hamster PCNA is not in a Our data are consistent with the by Bravo and Celis (14Bravo R. Celis J. FEBS Lett. 1985; 182: 435-440Crossref PubMed Scopus (34) Google Scholar) but in with that of Prosperi et al. (13Prosperi E. Scovassi A.I. Stivala L.A. Bianchi L. Exp. Cell Res. 1994; 215: 257-262Crossref PubMed Scopus (67) Google Scholar). PCNA in acetylation in the of functions T. J. 19: PubMed Scopus Google Scholar, A. 3: PubMed Scopus Google Scholar, T. 2001; PubMed Scopus Google Scholar, S. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google we PCNA acetylation. from the cells that with [3H]sodium acetate were by two-dimensional PAGE and a membrane, which was then to an for 4 or with PC10 Because the same was used to A and we were to PCNA the in To further this immunoprecipitated by PC10 were by two-dimensional by using anti-acetyl antibody or PC10 Consistent with the data from the in vivo A and this result that PCNA is we have that hamster PCNA is acetylated in the The PCNA in the three PCNA isoforms are of in acetylation were incubated with HDAC1 or TSA by two-dimensional of the sample with HDAC1 in an in the form whereas with TSA in an in the A and M forms with a in the form data that the A and M forms highly and moderately whereas the form is it was also that HDAC1 and did not or PCNA. be to of and deacetylase in the not all the acetylation were under our experimental conditions. Subcellular of PCNA in the of Cell Cycle PCNA protein in several subcellular at different cell cycle as described previously (15Naryzhny S.N. Lee H. Electrophoresis. 2001; 22: 1764-1775Crossref PubMed Scopus (40) Google Scholar, 17Spector D.L. Goldman R.D. Leinwand L.A. The Nuclear Matrix: Preparation for Microscopy and Biochemical. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 44.1-44.4Google Scholar, 18Ramsby M. Makowski G. Link A.J. 2-D Proteome Analysis Protocol. Humana Press Inc., Totowa, NJ1999: 53-66Google Scholar). shown in the of PCNA in the nuclear extract soluble nuclear chromatin and nuclear matrix were cell whereas of the cytosol fraction were the cell cycle The PCNA in and and the G1/S respectively and the most was in the fraction at the G1/S boundary data that PCNA to the nuclear matrix in and the nuclear to chromatin DNA replication We the PCNA acetylation status was different in each subcellular fraction. shown in the M form was found in the whereas of the A form were in the NEF, CMF, and Most the form was found in the fraction at a to that of the M Taken together, our data that the A and M forms of PCNA participate in the DNA replication process, whereas the form the was the of the A form was in the than To PCNA acetylation status is associated with cell cycle we the of the PCNA isoforms in the different cell cycle shown in cells in G0 by isoleucine starvation showed A and forms h of the A form is likely from the cell in in the cells in G0 were the cell cycle by them for h in of the cells were in in cells with the of the A and forms In contrast, most cells were in and of the A and forms were found the cells were incubated in the containing TSA MEM deacetylation was any A form was in the TSA-treated sample The data are also consistent with the that cells in G0 contained highly acetylated PCNA PCNA and be by p300 and HDAC, the that are responsible for the acetylation and deacetylation of PCNA, the immunoprecipitated by PC10 were to immunoblot analysis by using and shown in HDAC1 was co-immunoprecipitated with PCNA by PC10 that deacetylation of PCNA is by a with was also co-immunoprecipitated with PCNA this of data that PCNA be acetylated and deacetylated by p300 and respectively. is consistent with the in vitro data presented in 4 and The acetylation of PCNA by p300 be essential for DNA replication, are not of DNA S. R. 2001; PubMed Scopus Google Scholar). PCNA to DNA than a the acetylation status of PCNA ability to bind to DNA the immunoprecipitated by PC10 from cell treated by HDAC1 or TSA were to immunoblot analysis using anti-DNA polymerase PCNA treated with HDAC1 showed lower affinity to DNA polymerases β and δ than the sample treated with TSA 4 The of affinity was notable between deacetylated PCNA and DNA polymerase δ 4 and 5 of the has that PCNA acetylation and deacetylation an for to DNA polymerases for DNA replication. We carried out in vitro DNA replication assay after the PCNA complex immunoprecipitated by PC10 from was treated with TSA or shown in the sample treated with HDAC1 (i.e. showed lower DNA polymerization activity than that treated with data are consistent with the idea that PCNA acetylation is for DNA replication. together, our data support that PCNA in the M form to the replication that are associated with the nuclear matrix, in to be that the have at the of DNA replication by this H. S. Cell Biol. PubMed Scopus Google Scholar). also be that the replication by are associated and with the nuclear matrix P.A. Hamlin J.L. Mol. Cell. Biol. 8: PubMed Scopus Google Scholar). The PCNA chromatin replication also be the M form it is the form both in and The A form also be involved in DNA replication, in the of PCNA a of this form was found in the CMF, NMF, and but not in the fraction is to that a high of the form is found in the nuclear soluble fraction (i.e. NEF, Because the form did not show high affinity to DNA polymerases β and δ it not be involved in DNA replication. our data are consistent with the idea that the PCNA form is associated with replication our data not deacetylation the of PCNA from DNA polymerases and or PCNA is deacetylated after from chromatin. The of this PCNA by a of this and

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