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Nucleic Acids Research, 2003, Vol. 31, No. 17 5202-5211
© 2003 Oxford University Press

Phosphorylation of the PCNA binding domain of the large subunit of replication factor C on Thr506 by cyclin-dependent kinases regulates binding to PCNA

Isabelle Salles-Passador1, Anil Munshi2, Dominique Cannella1, Vincent Pennaneach1,2, Stephane Koundrioukoff1, Michel Jaquinod1, Eric Forest1, Vladimir Podust3, Arun Fotedar2 and Rati Fotedar*,1

1 Institut de Biologie Structurale, J.-P. Ebel, 41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France, 2 Sidney Kimmel Cancer Center, 10835 Altman Road, San Diego, CA 92121, USA and 3 Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA

*To whom correspondence should be addressed. Tel: +33 4 38 78 96 15; Fax: +33 4 38 78 54 94; Email: fotedar{at}ibs.fr
Correspondence may also be addressed to Arun Fotedar. Tel: +1 858 450 5990; Fax: +1 858 450 3251; Email: afotedar{at}skcc.org
Present address:
Stephane Koundrioukoff, Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich Irchel, Zurich, Switzerland

Received March 26, 2003; Revised June 10, 2003; Accepted June 30, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Replication factor C (RF-C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF-C (RF-Cp145) which binds to PCNA. We show here that the functional interaction of RF-Cp145 with PCNA is regulated by cdk-cyclin kinases. Phosphorylation of either RF-Cp145 as a part of the RF-C complex or RF-Cp145 domain B by cdk-cyclin kinases inhibits their ability to bind PCNA. A cdk-cyclin phosphorylation site, Thr506 in RF-Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr506->Ala RF-Cp145 domain B mutant is a poor in vitro substrate for cdk-cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr506 is mediated by CDKs since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk-cyclin phosphorylation site within the PCNA binding domain of RF-Cp145.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Replication of DNA is a strictly regulated event which occurs at a discrete period during the cell cycle. Cell cycle progression is regulated by a family of cyclin-dependent kinases (CDKs), which phosphorylate and activate proteins that execute events critical to cell cycle progression (reviewed in 1). In mammalian cells, the kinase activity associated with cdk4-cyclin D or cdk6-cyclin D is required for G1 progression (reviewed in 2). Cyclin E in association with cdk2 is required for the G1 to S phase transition, while cyclin A in complex with cdk2 functions later, for DNA replication and progression through S phase. The cdc2-cyclin B kinase in concert with cdk2-cyclin A controls events that promote entry into mitosis. Processive DNA synthesis by DNA polymerases {delta} and {epsilon} requires the cellular replication factor C (RF-C) and proliferating cell nuclear antigen (PCNA). RF-C binds to the 3' end of the RNA-DNA primer bound to template DNA and recruits PCNA to load it onto DNA in a process that requires ATP (reviewed in 3). PCNA forms a sliding clamp on DNA which then recruits DNA polymerases (reviewed in 4). The loading of PCNA onto DNA by RF-C is an important step in replication.

RF-C, a complex of five subunits, is conserved in all eukaryotes (reviewed in 5). In yeast, all subunits of RF-C are essential for viability. The genes encoding all five subunits of mammalian RF-C (145, 40, 38, 37 and 36 kDa) have been cloned (6–12). Functional homologs of RF-C exist in prokaryotes (T4 bacteriophage and Escherichia coli). Alignment of the subunits of RF-C from yeast and human with functionally homologous proteins from prokaryotes (T4 bacteriophage and E.coli) reveals short stretches of conserved amino acids, referred to as RF-C boxes (13).

We have previously shown that the large subunit of human RF-C (RF-Cp145, also referred to as hRF-Cp140) contains two tandem regions in the middle of the open reading frame which bind DNA and PCNA, respectively (12). The PCNA binding region, which we refer to as domain B, is important for RF-C activity, since a complex of RF-C with a deletion of domain B is unable to load PCNA onto DNA (14). We now find that phosphorylation of RF-C by cdk-cyclin kinases in vitro suppresses its ability to bind PCNA. The PCNA binding domain B of RF-Cp145 is also phosphorylated in vitro by cdk-cyclin kinases and this phosphorylation dramatically reduces its ability to bind PCNA. We have identified two sites, Thr506 and Ser518, in RF-Cp145 that are phosphorylated by cdk-cyclin kinases by mass spectrometry. By comparison of phosphopeptide maps of transiently transfected wild-type RF-Cp145 domain B with Thr506 and Ser518 site mutants, we have identified Thr506 as a phosphorylation target in mammalian cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Generation of recombinant fusion proteins
The cDNAs encoding domains A, B and A+B of the RF-Cp145 gene were cloned in-frame into pGEX vectors which express RF-Cp145 as a GST fusion protein (12). The Thr506->Ala mutant (Thr506Ala) and the Ser518->Ala mutant (Ser518Ala) were generated by PCR. Recombinant proteins were produced in E.coli (DH5{alpha} or BL21) as described earlier (12). Recombinant PCNA with an N-terminal His10 tag was purified from E.coli strain BL21(DE3) using a Ni2+-NTA column as described in Jonsson et al. (15). All recombinant proteins were dialyzed (12) and stored in aliquots at –80°C.

Immunoprecipitation with RF-Cp145 specific antibody
A polyclonal rabbit antiserum specific for RF-Cp145 was generated by immunizing rabbits with recombinant protein spanning amino acid residues 369–480 of RF-Cp145. The serum was pre-adsorbed to GST beads and then affinity purified on agarose affinity matrices covalently linked with the recombinant protein. In Figure 1, protein G–Sepharose (10 µl) was incubated with 1 µl of either anti-RF-Cp145 or preimmune antibody for 1 h at 4°C. After four washes with TNNE buffer (20 mM Tris, 250 mM NaCl, 0.5% NP-40, 5 mM EDTA), the beads were incubated with 300 µg MANCA cell extract (16). The mixture was adjusted to 250 mM NaCl, 0.5% NP-40, 5 mM EDTA. After 2 h at 4°C, the beads were washed twice with TKE buffer (20 mM Tris pH 7.5, 1% NP-40, 1 mM EDTA) and once with kinase buffer (40 mM HEPES pH 7.5, 8 mM MgCl2). The immunoprecipitate was then phosphorylated in an 18 µl standard kinase assay. After phosphorylation, the beads were washed twice with TKE buffer and once with kinase buffer. Binding with 0.1 µM His-PCNA was performed in HAMN buffer (40 mM HEPES pH 7.5, 1 mM ATP, 5 mM MgCl2, 300 mM NaCl) for 30 min. Beads washed with TNNE buffer were then analyzed for associated PCNA by western blotting with PCNA specific antibody. The anti-RF-Cp37 antibody kindly provided by Dr J. Hurwitz was used to verify that RF-C was immunoprecipitated as a complex by anti-RF-Cp145 antibody.



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  		Figure 1. Phosphorylation of RF-Cp145 by cdk-cyclin kinases in vitro reduces PCNA binding. (A) RF-C immunoprecipitated from cell extracts using RF-Cp145 specific antibody was western blotted with RF-Cp145 or RF-Cp37 specific antibodies (left). Note that both RF-Cp145 and RF-Cp37 are rabbit antibodies. Immunoprecipitated RF-C phosphorylated in vitro by cdc2-cyclin B kinase in the presence of [{gamma}-32P]ATP is shown on the right. The products of the kinase reaction were run on SDS–PAGE gels and the phosphorylation of proteins was analyzed by autoradiography. The position of RF-Cp145 is indicated. Histone H1 was used as a positive control substrate in a parallel kinase reaction. The masses of pre-stained molecular weight markers in kDa are indicated. (B) RF-Cp145 phosphorylated by cdc2-cyclin B as in (A) was incubated with His-PCNA. The immunoprecipitates were analyzed by western blotting using PCNA specific antibody. Phosphorylation of RF-Cp145 immunoprecipitates leads to reduced binding to PCNA as compared to a mock-phosphorylated control. As a control, cell extracts were immunoprecipitated with preimmune antibody (C) and then subject to phosphorylation with cdc2-cyclin B kinase. Note that a mouse PCNA antibody was utilized and hence it does not detect the antibody used for immunoprecipitation. Immunoprecipitated RF-Cp145 detected by RF-Cp145 specific antibody is also shown. Similar results were obtained in more than three independent experiments.

 
Cdk-cyclin kinase assays and phosphatase treatment
All kinase assays were performed with pre-activated cdk-cyclin kinases as described earlier (17). Briefly, individual cyclins, cdk2 and cdc2, were obtained from baculovirus infected Sf9 cells. Individual cyclins were mixed in appropriate amounts with cdk2 or cdc2 and incubated at 25°C for 20 min to obtain activation of kinase activity in vitro. Indicated amounts of GST fusion proteins were phosphorylated with pre-activated cdk-cyclin kinase in an 18 µl reaction mixture composed of 40 mM HEPES, 8 mM MgCl2, 166 mM ATP and 1 µCi [{gamma}-32P]ATP (3000 Ci/mmol; Amersham). After 30 min at 37°C, the reactions were stopped by adding SDS sample buffer. Mock phosphorylations were performed in the presence of kinase but without ATP. The reactions were run on 12% SDS–PAGE gels, the gels stained with Coomassie Brilliant Blue, dried and autoradiographed. One micromolar histone H1 (Fig. 2B) or GST–RF-Cp145 domains A, B or A+B (Fig. 2B and C) were used as substrates in an in vitro kinase assay.



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  		Figure 2. RF-Cp145 domain B is phosphorylated in vitro by cdk-cyclin kinases. (A) Schematic presentation of the putative cdk-cyclin kinase phosphorylation sites in the PCNA binding domain B of RF-Cp145. The consensus cdk phosphorylation motif is -S/T-P-X-Z, with X being a polar amino acid and Z a basic amino acid. The target residues in domain B are boxed. Domain A does not reveal any putative CDK phosphorylation sites. (B) Equal amounts of GST fusion proteins of RF-Cp145 domains A+B or domain B were phosphorylated in vitro by cdk2-cyclin A, cdk2-cyclin E and cdc2-cyclin B kinase. The products of the kinase reaction were run on SDS–PAGE gels and the phosphorylation of proteins was analyzed by autoradiography. Histone H1 was used as a positive control substrate in a parallel kinase reaction. Phosphorylation of H1, RF-C (A+B) and RF-C (B) by cyclin A-cdk2 kinase gave 90, 15 and 18 pmol phosphate incorporated, respectively; phosphorylation of H1, RF-C (A+B) and RF-C (B) by cdk2-cyclin E kinase gave 248, 38 and 45 pmol phosphate incorporated, respectively; phosphorylation of H1, RF-C (A+B) and RF-C (B) by cdc2-cyclin B kinase gave 540, 108 and 90 pmol phosphate incorporated, respectively. Phosphorylation of RF-C (A+B) and RF-C (B) with all three kinases was 15–20% of that obtained with histone H1. We generally load one-sixth of the amount of histone H1 kinase reaction. Similar results were obtained in six independent experiments. (C) GST fusion protein of RF-Cp145 domain A is not phosphorylated in vitro by cdc2-cyclin B kinase. The left panel shows the autoradiograph and the right panel shows a Coomassie Blue stained gel of the two proteins used as substrates in the in vitro kinase reaction.

 
In Figure 6A, cdc2-cyclin B phosphorylated RF-Cp145 was dephosphorylated by incubating the kinase reaction mix with 20 U alkaline phosphatase (Roche Biochemicals) in a 25 µl reaction with 1x final concentration of the buffer provided with the phosphatase. The reaction was carried out for 30 min at 37°C.



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  		Figure 6. Phosphorylation of endogenous RF-Cp145 at Thr506 by CDKs. (A) Anti-phospho-Thr506 RF-Cp145 antibody specifically recognizes phosphorylated Thr506 in RF-Cp145 domain B. Equal amounts of GST fusion proteins of RF-Cp145 domains A+B, domain B and Thr506 Ala domain B mutant were phosphorylated in vitro by cdc2-cyclin B kinase. The products of the kinase reaction were run on SDS–PAGE gels and western blotted with anti-phospho-Thr506 RF-Cp145 antibody. Only phosphorylated RF-Cp145 domains A+B and domain B are recognized by the antibody (left). Anti-phospho-Thr506 RF-Cp145 antibody does not recognize phosphorylated RF-Cp145 Thr506Ala domain B mutant. Dephosphorylation of cdc2-cyclin B phosphorylated RF-Cp145 domain B with alkaline phosphatase (see Materials and Methods) results in loss of the reactivity with anti-phospho-Thr506 RF-Cp145 antibody (right). (B) Anti-phospho-Thr506 RF-Cp145 antibody immunoprecipitates the phosphorylated form of RF-Cp145 from HeLa cell extracts. HeLa cell extract immunoprecipitated with either anti-phospho-Thr506 or with anti-RF-Cp145 antibodies were analyzed by western blotting with RF-Cp145 antibody. (C) Phosphorylation of Thr506 in RF-Cp145 depends on CDKs. HeLa cells were treated with roscovitine for 2 h prior to making cells extracts. Treatment with roscovitine resulted in a substantial loss of Thr506 phosphorylation.

 
Phosphorylation of RF-Cp145 and binding to PCNA
For Figure 3A, 1.2 µM GST–RF-Cp145 was phosphorylated with pre-activated cdk-cyclin kinase in a standard kinase assay except that [{gamma}-32P]ATP was omitted from reactions that were subsequently analyzed by western blotting. A control tube with [{gamma}-32P]ATP in parallel was included to verify phosphorylation of GST–RF-Cp145 proteins. After phosphorylation, 1 µM His-PCNA was added and the mix incubated for 30 min at 25°C (12). The complexes were collected by incubating with 10 µl glutathione–Sepharose beads for 1 h at 4°C. After 3 washes in TENK buffer (20 mM Tris pH 8, 1 mM EDTA, 100 mM KCl, 0.5% NP-40), the PCNA bound to RF-Cp145 on glutathione–Sepharose was visualized by western blotting with PCNA specific antibody.



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  		Figure 3. Phosphorylation by cdk-cyclin kinases inhibits the ability of RF-Cp145 domain B to bind PCNA but not DNA. (A) GST fusion protein of RF-Cp145 domains A+B was phosphorylated by cdc2-cyclin B, cdk2-cyclin A or by cdk2-cyclin E kinases and then tested for its ability to bind PCNA. We verified that equal amounts of RF-Cp145 were bound to beads. Phosphorylation of domain B leads to reduced binding of RF-Cp145 domains A+B to PCNA. Similar results were obtained in more than three independent experiments. (B) Phosphorylation of RF-Cp145 domains A+B by cdk-cyclin kinases is reduced in the presence of PCNA. The ability of cdc2-cyclin B kinase to phosphorylate 6 µM domains A+B in the presence of increasing amounts of His-PCNA (0.5, 1, 5 and 10 µM) was determined. The products of the kinase reaction were loaded on SDS–PAGE gels and phosphorylation of RF-Cp145 analyzed by autoradiography. (C) Phosphorylation of RF-Cp145 domains A+B by cdc2-cyclin B kinases is unaffected in the presence of DNA. RF-Cp145 domains A+B (10 µM) bound to glutathione–Sepharose was incubated with increasing amounts of M13 primed DNA (4, 8 and 16 µg/µl) in the presence of [{gamma}-32P]ATP. After washing, the glutathione–Sepharose bound RF-Cp145 domains A+B was phosphorylated by cdc2-cyclin B kinase. The products of the kinase reaction were loaded on SDS–PAGE gels and phosphorylation of RF-Cp145 analyzed by autoradiography. (D) Phosphorylated RF-Cp145 domains A+B bound to dsDNA–cellulose shows a significantly reduced ability to bind PCNA. RF-Cp145 domains A+B bound to dsDNA–cellulose was phosphorylated with cdc2-cyclin B kinase and then incubated with PCNA. Ability to bind PCNA was analyzed by western blotting using PCNA antibody. PCNA alone, which does not bind to dsDNA–cellulose in the absence of RF-Cp145, is shown as a control (right). Similar results were obtained in three independent experiments. (E) cdk-cyclin kinase phosphorylated RF-Cp145 domain B fails to inhibit in vitro DNA replication. Domain B immobilized on glutathione–Sepharose beads was either mock-phosphorylated or phosphorylated with cdc2-cyclin B kinase. The cdc2-cyclin B kinase was washed off after phosphorylation and the effect of phosphorylated domain B on in vitro DNA replication determined. In the left panel, three different concentrations of phosphorylated domain B and mock-treated domain B were tested in SV40 origin-dependent in vitro replication assays. The right panel shows that three different preparations of phosphorylated RF-Cp145 domain B (at 2 µM) fail to inhibit DNA replication as compared to 2 µM control domain B.

 
For Figure 3D, 1.2 µM GST–RF-Cp145 was incubated with 10 µl double-stranded DNA (dsDNA)–cellulose in 30 µl buffer containing 20 mM Tris pH 8, 1 mM EDTA, 10 mM NaCl and 1 mM DTT for 1 h at 4°C. After three washes in TENK buffer, immobilized RF-Cp145 protein was phosphorylated with pre-activated cdc2-cyclin B kinase and incubated with His-PCNA for 30 min at 25°C. After three washes in TENK buffer, PCNA bound to RF-Cp145 was visualized by western blotting with PCNA specific antibody.

Phosphorylation of RF-Cp145 in the presence of primed M13 DNA or PCNA
In Figure 3B, 1.2 µM GST–RF-Cp145 was incubated with various concentrations of His-PCNA in kinase buffer for 30 min at 25°C and with 10 µl dsDNA–cellulose for a further 1 h at 4°C. After three washes in TENK, the bound RF-Cp145 proteins were subjected to phosphorylation by cdk-cyclin kinase.

In Figure 3C, M13 DNA (Promega) diluted to 0.1 mg/ml in annealing buffer (10 mM Tris pH 8, 2.5 mM MgCl2, 125 mM NaCl) was mixed with 0.25 µM of 24mer oligonucleotide primer. The mixture was heated at 75°C for 15 min and the primer and the single-stranded M13 DNA were annealed slowly at room temperature. The primed M13 DNA was then incubated with glutathione–Sepharose bound GST–RF-Cp145 proteins. After 15 min the beads were washed and the glutathione–Sepharose bound RF-Cp145 proteins were subjected to phosphorylation by cdk-cyclin kinase.

Cell labeling, immunoprecipitation and peptide maps
Cos cells were transiently transfected with vectors expressing HA epitope tagged RF-Cp145 domain B (12) or Thr506Ala domain B point mutant or Ser518Ala domain B point mutant. Forty-eight hours after transfection cells were washed twice with HEPES-buffered saline and incubated for 2–4 h with phosphate-free Dulbecco’s modified Eagle’s medium (DMEM) containing 5% Calf bovine serum and 1 mCi [32P]ortho phosphate (ICN 64013) at 106 cells/ml. Cell extracts were prepared in a buffer containing 50 mM HEPES (pH 7.5), 0.1% Triton X-100, 0.5 M NaCl, 5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM NaF and 1 mM sodium vanadate. The 32P-labeled RF-Cp145 domain B was immunoprecipitated with an anti-HA epitope specific monoclonal antibody (BabCo). The immune complexes were precipitated overnight at 4°C with protein G–Sepharose. The proteins were eluted by boiling beads in sample buffer, resolved on a 10% denaturing polyacrylamide gel and electroblotted to nitrocellulose. The 32P-labeled domain B protein was visualized by autoradiography and the corresponding piece of nitrocellulose excised. Mapping of the phosphate labeled tryptic peptides was performed as described previously (18). Nitrocellulose membrane pieces were blocked with polyvinylpyrrolidone 360 (0.5% in 100 mM acetic acid), washed with 50 mM ammonium bicarbonate and then subjected to trypsin digestion (three times with 10 mg of TPCK treated trypsin for 16 h at 37°C). Eluted phosphopeptides were resolved on thin-layer chromatography plates by electrophoresis for 27 min at 1000 V in pH 4.72 buffer (5% n-butanol, 2.5% glacial acetic acid and 2.5% pyridine) in the first dimension (anode on left and cathode on right) using an HTLE-7000 apparatus (CBS Scientific, Del Mar, CA), followed by ascending chromatography in isobutyric acid chromatography buffer (62.5% isobutyric acid, 1.9% n-butanol, 4.8% pyridine and 2.9% glacial acetic acid) in the second dimension. The phosphorylated peptides were visualized by autoradiography and phosphorimager analysis.

Mapping of in vitro phosphorylation sites by mass spectrometry
RF-Cp145 domain B GST fusion protein immobilized on glutathione–Sepharose beads was phosphorylated with cdc2-cyclin B kinase using 32P-labeled ATP. The phosphorylated and mock-phosphorylated domain B proteins were eluted and then digested with endoproteinase Lys-C (50 mM NH4HCO3 pH 7.2 for 2 h at 37°C, enzyme:substrate ratio 1:20 w/w). Immobilized recombinant RF-Cp145 domain B GST fusion protein phosphorylated with cdc2-cyclin B kinase using non-radioactive ATP was digested with endoproteinase Glu-C (50 mM NH4HCO3 pH 7.2 for 12 h at 35°C, enzyme:substrate ratio 1:100 w/w). The reactions were stopped by cooling the sample to –20°C. The peptides obtained after digestion of phosphorylated and non-phosphorylated RF-Cp145 were separated by reverse phase high performance liquid chromatography (Applied Biosystems 130A) using a reverse phase column (Brownlee, C18, 5 µm, 1 x 100 mm). Solvent A was a solution of 0.1% trifluoroacetic acid in water and solvent B was a 90:10:0.08 acetonitrile:water:trifluoroacetic acid mixture. The column was equilibrated in 0% solvent B and the peptides were separated with a gradient of solvent B (0–50% in 40 min) at a flow rate of 50 µl/min. The collected fractions were concentrated.

Mass spectra were obtained with a MALDI time-of-flight mass spectrometer (Voyager Elite Xl; Perseptive Biosystems). All spectra were acquired in the positive ion mode with an acceleration voltage of 20–25 kV. Aliquots of 0.5 µl of the peptide solution and 0.5 µl of the matrix solution were mixed on the stainless steel sample plate and dried prior to MS analysis. The matrix was a saturated solution of 2.5 dihydroxybenzoic acid prepared in a 50% (v/v) solution of acetonitrile:0.1% trifluoroacetic acid in water. External calibration was performed with bovine insulin (Sigma).

The calculated masses of the peptides obtained after proteolysis of RF-Cp145 domain B were obtained using the amino acid sequence of the protein and the MacBioSpec software (Perkin-Elmer).

In vitro DNA replication assays
For Figure 3E, GST–RF-Cp145 was phosphorylated with cdk-cyclin kinase, prior to elution with glutathione containing buffer as described earlier (12). The eluate containing the phosphorylated protein was equilibrated with buffer (4 mM HEPES pH 7.5, 1 mM MgCl2, 1 mM DTT, 10% glycerol) and concentrated prior to use in replication reactions. Replication reactions containing 150 ng of SV40 origin containing plasmid DNA, with 1 µg of SV40 T antigen and 100 µg of S100 extract from S phase Manca cells, were performed as described (12,19). The amount of DNA synthesis was quantitated by measuring the incorporation of [{alpha}-32P]dCTP into trichloroacetic acid precipitable counts.

Generation of phospho site specific antibody
Rabbits were immunized with human RF-Cp145 phosphorylated peptide [KESKLER(T-PO4)PQKNVQGC]. The phospho site specific antibodies were affinity purified using a column of the phosphorylated peptide as antigen. The affinity purified anti-phospho-Thr506 antibodies were further purified through a column of the non-phosphorylated peptide.

Cell extract preparation and immunoprecipitation with phospho site specific antibody
HeLa cells were grown in DMEM plus 10% bovine serum. For preparation of extracts, cells washed with phosphate-buffered saline were scraped into a buffer containing 10 mM HEPES, 0.1% Triton X-100, 0.5 mM dithiothreitol (DTT), 1.5 mM MgCl2, 10 mM KCl, 2 µg/ml each aprotinin and leupeptin, 0.5 mM PMSF, 50 mM sodium fluoride, 1 mM sodium orthovanadate. After 10 min at 4°C, the cells were spun at 10 000 g for 10 min. The supernatant represents soluble cytoplasmic components. The pellet was resuspended in a buffer containing 50 mM HEPES, 0.1% Triton X-100, 0.5 mM DTT, 5 mM EDTA, 0.5 M NaCl, 2 µg/ml each aprotinin and leupeptin, 0.5 mM PMSF, 50 mM sodium fluoride, 1 mM sodium orthovanadate. After centrifugation for 10 min at 4°C, the supernatant which includes the tight nuclear associated proteins (20) was collected and used for western blot analysis and for immunoprecipitation. Where indicated, cells were treated with 50 µM roscovitine (Sigma) diluted in DMSO for 2 h prior to cell extract preparation. In Figure 6B, 1 mg cell extract was immunoprecipitated with 20 µl of protein G–Sepharose preincubated with 3 µl of either polyclonal RF-Cp145 antibody or anti-phospho-Thr506 antibody. After 2 h (or overnight) at 4°C, the beads were washed three times with TNE buffer (20 mM Tris, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 2 µg/ml each aprotinin and leupeptin, 1 mM PMSF, 50 mM sodium fluoride) before analyzing the pellet by western blotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
RF-Cp145 domain B is phosphorylated in vitro by cdk-cyclin kinases
During the course of our work on the effect of CDKs on the RF-C complex, we noticed that CDK phosphorylation suppressed the ability of RF-C to bind PCNA. RF-C immunoprecipitated from cell extracts with RF-Cp145 specific antibody was phosphorylated with CDKs. The small subunits are associated with the large subunit of RF-C as suggested by the presence of associated RF-Cp37 (Fig. 1A, left panel) in the RF-Cp145 immunoprecipitates. Only the large subunit of RF-C (RF-Cp145) was phosphorylated by cdc2-cyclin B kinase (Fig. 1A, right panel). The small subunits are not phosphorylated. This phosphorylated RF-C was then examined for its ability to bind His-PCNA in vitro. Under these conditions, after phosphorylation, significantly less PCNA was associated with the immobilized RF-C immunoprecipitates as compared to mock-phosphorylated immobilized RF-C (Fig. 1B).

We have previously shown that the large subunit of human RF-C (RF-Cp145) contains a PCNA binding region referred to as domain B (12), which is important for RF-C activity (14). The sequence of RF-Cp145 revealed two putative CDK phosphorylation sites, Thr506 and Ser518, in the PCNA binding domain B (Fig. 2A). The consensus motif for phosphorylation by cdk2/cdc2 kinases is -S/T-P-X-Z, with X being a polar amino acid and Z a basic amino acid, and it differs from that for phosphorylation by cdk4-cyclin D1 kinase (21). To determine whether the PCNA binding domain B was phosphorylated by cdk-cyclin kinases, GST fusion proteins of RF-Cp145 domains A+B (residues 395–728), A (residues 395–480) and B (residues 480–728) (Fig. 2A) were produced in bacteria and purified by glutathione–Sepharose affinity chromatography. The purified proteins were then subjected to in vitro phosphorylation with cdk2-cyclin A, cdk2-cyclin E or cdc2-cyclin B kinase and phosphorylation of products was examined by autoradiography following separation by SDS–PAGE. The result from one representative experiment with RF-Cp145 domains A+B and RF-Cp145 domain B is shown in Figure 2B. Both RF-Cp145 domains A+B and domain B were phosphorylated by cdk-cyclin kinases in vitro. In contrast to RF-Cp145 domain B, the GST fusion protein of domain A was not phosphorylated by cdk-cyclin kinases (Fig. 2C), indicating that domain A has no CDK phosphorylation sites.

Phosphorylation of RF-Cp145 domain B prevents binding to PCNA but not to DNA
We then tested whether phosphorylation of domain B by cdk-cyclin kinases interferes with its ability to bind PCNA. For this, GST–RF-Cp145 domains A+B was phosphorylated with cdk2-cyclin A, cdk2-cyclin E or cdc2-cyclin B kinase and then tested for its ability to bind to PCNA. The result shows that phosphorylation of RF-Cp145 domains A+B uniformly results in reduced binding to PCNA (Fig. 3A).

We also determined the ability of cdk-cyclin kinase to phosphorylate domain B in the presence of increasing amounts of purified PCNA (Fig. 3B). Phosphorylation of RF-Cp145 domains A+B by cdk-cyclin kinases was reduced to less than 50% at an approximately 1:1 molar ratio of PCNA (5 µM) to domains A+B. Similar results were obtained with RF-Cp145 domain B (data not shown). These results suggest that phosphorylation by cdk-cyclin kinases and binding by PCNA utilize overlapping if not identical regions of RF-Cp145 domain B.

Phosphorylation of RF-Cp145 domains A+B by cdk-cyclin kinases was unaffected by the presence of dsDNA as determined by the ability of cdk-cyclin kinase to phosphorylate residues in domain B of RF-Cp145 in the presence of an increasing molar ratio of dsDNA in the form of M13 primed DNA (Fig. 3C). Consistent with this, phosphorylation of RF-Cp145 domains A+B bound to DNA interferes with its ability to bind PCNA. GST–RF-Cp145 domains A+B bound to dsDNA–cellulose was phosphorylated with cdk-cyclin kinase and then tested for its ability to bind to PCNA. Under these conditions, phosphorylation of RF-Cp145 domains A+B leads to a significant reduction in its ability to bind PCNA (Fig. 3D). As expected, PCNA did not bind to dsDNA–cellulose in the absence of RF-Cp145 (Fig. 3D).

We then tested whether cdk-cyclin phosphorylated RF-Cp145 domain B could inhibit SV40 origin-dependent DNA replication in S phase extracts. RF-Cp145 domain B inhibits RF-C function in vitro through its ability to sequester PCNA, thereby decreasing the amount of PCNA available for DNA replication (12). For this assay, RF-Cp145 domain B immobilized on glutathione–Sepharose beads was phosphorylated with cdc2-cyclin B kinase. After washing the beads, the ability of phosphorylated domain B to inhibit DNA replication was tested. Phosphorylation by cdk-cyclin kinase considerably reduced the ability of domain B to inhibit DNA replication in a dose-dependent manner (Fig. 3E, left panel). Three different preparations of domain B assayed at 2 µM showed a similar failure to inhibit DNA synthesis after phosphorylation (Fig. 3E, right panel). We conclude that phosphorylation reduces the ability of domain B to bind PCNA.

Mapping of in vitro phosphorylation sites in domain B by mass spectrometry
The amino acid residues in PCNA binding domain B that are phosphorylated by cdk-cyclin kinases were mapped using a MALDI-TOF mass spectrometer. For this analysis, immobilized recombinant GST–RF-Cp145 domain B was phosphorylated with cdc2-cyclin B kinase using 32P-labeled ATP. Phosphorylated domain B protein was eluted and then digested with endoproteinase Lys-C. The resulting peptides were separated by reverse phase HPLC using a C18 column. Two 32P-labeled fractions were obtained and analyzed by mass spectrometry. Each radiolabeled fraction contained one peptide with 80 Da mass increase indicative of one phosphate per peptide (Table 1). The phosphopeptides identified are ESKLERT506PQK and RKIS518PSK.


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  		Table 1. RF-Cp145 phosphopeptides identified by mass spectrometry

 
Digestion of non-radiolabeled phosphorylated RF-Cp145 domain B with endoproteinase Glu-C followed by reverse phase HPLC and mass spectrometry enabled the identification of phosphorylated peptides (with no, one or two 80 Da mass increase) that cover the region containing the two putative cdk-cyclin phosphorylation sites (Table 1). Together these results confirm the presence of two cdk-cyclin phosphorylation sites, residues Thr506 and Ser518.

In vivo phosphorylation of RF-Cp145 domain B
To determine if the RF-Cp145 domain B is phosphorylated in vivo, Cos-7 cells were transiently transfected with vector expressing HA epitope tagged domain B and then labeled with [32P]orthophosphate. Extracts prepared from transfected cells after in vivo labeling were immunoprecipitated with HA specific antibody and the 2-dimensional phosphopeptide maps were analyzed after trypsin digestion. The results obtained demonstrate that PCNA binding domain B is phosphorylated in vivo (Fig. 4A). We then determined if the in vitro cdk-cyclin phosphorylation sites identified by mass spectrometry were also phosphorylated in vivo. For this, domain B expression vectors mutated at the specific phosphorylation sites we had mapped by mass spectrometry were generated. We then compared the 2-dimensional tryptic maps of transfected wild-type domain B (Fig. 4A) with a domain B point mutant in which Thr506 is mutated to Ala (Fig. 4B) and another in which Ser518 is mutated to Ala (Fig. 4C). Several major domain B phosphopeptides were absent in the 2-dimensional tryptic map of Thr506Ala domain B mutant. In contrast, analysis of the Ser518Ala domain B mutant revealed no change from wild-type domain B. These results allow us to conclude that at least Thr506 in domain B, which is phosphorylated by cdk-cyclin kinases in vitro, is a major in vivo phosphorylation site.



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  		Figure 4. Mapping of RF-Cp145 Thr506 as an in vivo phosphorylation site in mammalian cells. Cos-7 cells were transiently transfected with vectors expressing HA epitope tagged domain B, domain B in which Thr506 is mutated to Ala and domain B in which Ser518 is mutated to Ala. The cells were labeled with [32P]orthophosphate for 2 h prior to making extracts. Extracts were immunoprecipitated with HA specific antibody. The 2-dimensional tryptic maps of transfected wild-type domain B (left), Thr506 Ala domain B (middle) and Ser518Ala domain B (right) are shown. Several major phosphopeptides that were present in domain B were absent in the 2-dimensional tryptic map of Thr506Ala domain B but not of Ser518Ala domain B.

 
In order to confirm that Thr506 was indeed phosphorylated in vitro by cdk-cyclin kinases, we generated a GST fusion protein of the Thr506Ala RF-Cp145 domain B mutant. When the ability of cdk-cyclin kinases to phosphorylate RF-Cp145 domain B was compared to the RF-Cp145 Thr506Ala domain B mutant (Fig. 5A), we observed a significant reduction in the phosphorylation of the Thr506Ala domain B mutant. In contrast, phosphorylation of a mutant in which irrelevant Thr and Ser were mutated to Ala (T532A S533A) was unaffected. We then tested the ability of the Thr506Ala domain B mutant to bind PCNA after phosphorylation by cdk-cyclin kinase. Phosphorylation did not alter the ability of the Thr506Ala domain B mutant to bind PCNA, whereas in the same experiment phosphorylation of wild-type domain B dramatically reduces the ability to bind PCNA (Fig. 5B).



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  		Figure 5. The Thr506Ala RF-Cp145 domain B mutant is a poor target for cdk-cyclin phosphorylation and retains the ability to bind PCNA after phosphorylation. (A) The comparative ability of GST fusion proteins of domain B and Thr506Ala domain B mutant to become phosphorylated by cdk2-cyclin A kinase was determined in an in vitro kinase assay in the presence of [{gamma}-32P]ATP. The products of the kinase reaction were loaded on SDS–PAGE gels and phosphorylation of RF-Cp145 analyzed by autoradiography. Phosphorylation of domain B mutant T532A S533A, in which an irrelevant threonine and serine are mutated, is shown as a control. The top panel shows the autoradiograph and the bottom panel shows a Coomassie Blue stained gel of proteins used as substrates in the kinase reaction. The position of the RF-Cp145 domain B proteins is indicated by a dot. (B) Phosphorylation of Thr506Ala domain B mutant by cdk-cyclin kinase does not lead to reduced binding to PCNA. Recombinant GST fusion proteins of wild-type domain B and Thr506Ala domain B mutant were phosphorylated by cdk2-cyclin A kinase and then tested for its ability to bind PCNA. The complexes were collected by binding to glutathione–Sepharose and analyzed by western blotting using PCNA specific antibody.

 
In vivo phosphorylation of endogenous RF-Cp145 at Thr506
To determine whether Thr506 is a phosphorylation site for endogenous RF-Cp145, polyclonal rabbit anti-phospho-Thr506 antibodies were raised against a RF-Cp145 peptide containing Thr506. Results obtained with ELISA indicated that the anti-phospho-Thr506 RF-Cp145 antibody recognizes the RF-Cp145 peptide containing phosphorylated Thr506 but not non- phosphorylated Thr506 (data not shown). GST fusion proteins of RF-Cp145 domain B, the Thr506Ala domain B mutant and RF-Cp145 domains A+B were phosphorylated in vitro with cdc2-cyclin B kinase and immunoblotted with anti-phospho-Thr506 RF-Cp145 antibody. Figure 6A (left panel) shows that anti-phospho-Thr506 RF-Cp145 antibody recognizes phosphorylated but not non-phosphorylated RF-Cp145 domain B and RF-Cp145 domains A+B. In particular, anti-phospho-Thr506 RF-Cp145 antibody does not recognize phosphorylated RF-Cp145 Thr506Ala domain B mutant. It should be noted that Ser518 is intact in this mutant, indicating that the antibody is highly specific for Thr506. Dephosphorylation of cdc2-cyclin B phosphorylated RF-Cp145 domain B with alkaline phosphatase results in loss of the reactivity with anti-phospho-Thr506 RF-Cp145 antibody (Fig. 6A, right panel). Further, phosphorylation of RF-C complex by cdc2-cyclin B kinase leads to phosphorylation of Thr506 in RF-Cp145 (see Supplementary Material Figure S2). Recombinant RF-C complex was generated by co-infection of insect cells with baculovirus encoding the five RF-C subunits (p140, p40, p38, p37 and p36) and purification on Ni-NTA and Mono-Q columns.

Anti-phospho-Thr506 RF-Cp145 antibody immunoprecipitates the phosphorylated form of RF-Cp145 from HeLa cell extracts (Fig. 6B). To determine whether RF-Cp145 recognition by anti-phospho-Thr506 RF-Cp145 antibody is a result of phosphorylation by cyclin-dependent kinases, cells were treated with roscovitine (22) prior to making cell extracts at the indicated times. Treatment with roscovitine resulted in a substantial loss of Thr506 phosphorylation (Fig. 6C). Taken together, these results show phosphorylation of Thr506 in endogenous RF-Cp145 and further that Thr506 is phosphorylated by cyclin-dependent kinases.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY MATERIAL
 REFERENCES
 
In this paper we show that phosphorylation of the PCNA binding domain B in RF-Cp145 by cdk-cyclin kinase interferes with its ability to bind PCNA. Although two cdk-cyclin kinase phosphorylation sites, Thr506 and Ser518, were identified in vitro in domain B by mass spectrometry, only Thr506 is phosphorylated in vivo in human cells. Using an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that Thr506 in endogenous RF-Cp145 is phosphorylated. This phosphorylation site is conserved among human, mouse and rat RF-Cp145. Unlike wild-type domain B, domain B with a Thr506->Ala substitution was a poor in vitro substrate for cdk-cyclin kinase. It is worth noting that phosphopeptide maps of RF-Cp145 domain B display different signals (Fig. 4). This result is best explained by phosphorylation of this region of domain B by other kinases besides CDKs. Since several major domain B phosphopeptides were absent in the 2-dimensional tryptic map of the Thr506Ala domain B mutant, it suggests that phosphorylation at Thr506 precedes phosphorylation by other kinases. In contrast, analysis of the Ser518Ala domain B mutant revealed no change from wild-type domain B.

We find that RF-C immunoprecipitated from cell extracts with RF-Cp145 specific antibody can be phosphorylated by CDKs in vitro. The large subunit of RF-C (RF-Cp145) was phosphorylated by CDKs. When phosphorylated immunoprecipitated RF-C was then examined for its ability to bind PCNA in vitro, it bound significantly less PCNA as compared to a mock-phosphorylated control. Further, phosphorylation by CDKs decreases the ability of RF-Cp145 domains A+B to bind PCNA. In vitro phosphorylation of domains A+B by CAMK II has a similar effect (23). Our study for the first time maps an in vivo phosphorylation site in RF-Cp145. Since the sites phosphorylated by Ca2+/calmodulin-dependent kinase II in domain B have not been mapped, future studies which map CAMK II phosphorylation sites will allow assessment of the relative functional roles of CAMK II and cdk-cyclin kinases in regulating RF-C activity.

It has been proposed that the large subunit initiates binding to PCNA, which is then stabilized by multiple interactions of PCNA within the RF-C complex (reviewed in 24). Although the N-terminus of RF-Cp145 harbors a PCNA binding site (25), the PCNA binding site in domain B is essential for RF-C function (14). The {gamma} complex in E.coli, like the yeast and human RF-C complexes, is comprised of five polypeptides ({delta}'-{gamma}1-{gamma}2-{gamma}3-{delta}) and loads the E.coli ß protein, the PCNA homolog, onto DNA for processive DNA replication. The structure of the {gamma} complex revealed that the C-terminal domains of {delta}', {gamma} and {delta} form a helical scaffold (circular collar) while the N-terminal ends appear to dangle under the C-terminal pentamer umbrella (26). The structure of a complex of the {delta} subunit with the ß (PCNA homolog) subunit (27) further reveals that the region in {delta} which contacts ß is homologous to domain B in human RF-Cp145. Especially relevant are the two hydrophobic residues Leu73 and Phe74 in {delta} that contact ß and appear to be critical for stabilizing the {delta}–ß interaction (27). The corresponding residues in human RF-Cp145 are Phe701 and Tyr702, and they are included in domain B. These residues are conserved from yeast to man. It is tempting to speculate that phosphorylation at Thr506 in RF-Cp145 induces a conformational change in RF-C which precludes the contact residues from binding PCNA. Structural studies with RF-C complex will help resolve this issue.

The CDK phosphorylated purified RF-C complex was slightly inhibitory (30%) as compared to the non-phosphorylated RF-C (data not shown) when assayed for PCNA loading using the in vitro assay (28). Phosphorylation of RPA-32, one of the three subunits of mammalian single-stranded DNA binding protein RPA, occurs during G1 to S phase (29). Phosphorylation of RPA-32, however, has no effect on RPA function in in vitro assays of binding to single-stranded DNA, DNA replication or DNA repair (30–33). In vivo, phosphorylated RPA-32 displays changes in subunit association (34). To understand the regulation of RF-C by cdk-cyclin kinases, further studies on the phosphorylation status of RF-C in vivo using antibodies that specifically recognize the phospho site combined with the use of specific inhibitors of cdk-cyclin kinase will be necessary.

Previous studies have shown that cdk-cyclin kinases regulate biological activity of their substrates by either influencing subcellular localization, altering rates of protein degradation and/or modulating binding to other proteins. Phosphorylation of Schizosaccharomyces pombe Cdc18 (35–37) and cdt1 (38) by cdk-cyclin kinases regulates their degradation. cdk-cyclin kinases trigger a switch in the subcellular localization of ScMCM4 (39), ScCdt1 (40) and hsCdc6 (41,42). hsCdc6 is a human homolog of Saccharomyces cerevisiae Cdc6 and S.pombe Cdc18. Cdc6 and cdt1 are essential for proper loading of MCMs onto DNA. Cdk-cyclin kinases regulate the activity of mammalian p68 subunit DNA polymerase {alpha} (43,44). Cdk-cyclin kinases are thus a critical component of the signaling pathways that regulate the function of replication proteins in the cell cycle.

Our study provides a first step towards understanding the functional regulation of RF-C by cyclin-dependent kinases. Using an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr506 is mediated by CDKs, since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. Phosphorylation of RF-Cp145 by CDKs may be regulated in a cell cycle-dependent manner. We find that kinase activity from mitotic extracts is most efficient in phosphorylating a GST fusion protein of RF-Cp145 domain B (unpublished observations). This kinase activity in mitotic extracts was inhibited by roscovitine. In fission yeast, mitotic cyclin-dependent kinases act locally at the replication origins to inhibit formation of pre-replication components (45). We and others have earlier reported that CDKs are associated with the mammalian replication complex (46–50). It is possible that CDKs associated with the mammalian replication complex phosphorylate and regulate the function of RF-C and perhaps other replication proteins.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at NAR Online.


   ACKNOWLEDGEMENTS
 
The authors acknowledge the kind gift of antibody recognizing RF-Cp37 by J. Hurwitz (Memorial Sloan-Kettering Cancer Center, New York, NY), His-PCNA plasmid by U. Hubscher (Universitat Zurich-Irchel, Switzerland) and recombinant baculovirus by David Morgan (University of California, San Francisco, CA). During the course of this work, I.S.-P. was the recipient of a fellowship from Region Rhône Alpes and V.P. was the recipient of a studentship from l’Association pour la Recherche sur le Cancer (ARC). This work was supported by grants to R.F. from the Region Rhône Alpes and ARC and by grants to A.F. from the National Institutes of Health (CA74435 and CA92321).


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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