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Originally published In Press as doi:10.1074/jbc.M710313200 on April 8, 2008

J. Biol. Chem., Vol. 283, Issue 23, 16104-16114, June 6, 2008
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Protein Phosphatase 2A Is Targeted to Cell Division Control Protein 6 by a Calcium-binding Regulatory Subunit*Formula

Anthony J. Davis{ddagger}1, Zhen Yan§, Bobbie Martinez{ddagger}, and Marc C. Mumby{ddagger}2

From the {ddagger}Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041 and §the Division of Cardiology, Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710

Received for publication, December 19, 2007 , and in revised form, February 29, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cell division control protein 6 (Cdc6) is essential for formation of pre-replication complexes at origins of DNA replication. Phosphorylation of Cdc6 by cyclin-dependent kinases inhibits ubiquitination of Cdc6 by APC/Ccdh1 and degradation by the proteasome. Experiments described here show that the PR70 member of the PPP2R3 family of regulatory subunits targets protein phosphatase 2A (PP2A) to Cdc6. Interaction with Cdc6 is mediated by residues within the C terminus of PR70, whereas interaction with PP2A requires N-terminal sequences conserved within the PPP2R3 family. Two functional EF-hand calcium-binding motifs mediate a calcium-enhanced interaction of PR70 with PP2A. Calcium has no effect on the interaction of PR70 with Cdc6 but enhances the association of PP2A with Cdc6 through its effects on PR70. Knockdown of PR70 by RNA interference results in an accumulation of endogenous and expressed Cdc6 protein that is dependent on the cyclin-dependent protein kinase phosphorylation sites on Cdc6. Knockdown of PR70 also causes G1 arrest, suggesting that PR70 function is critical for progression into S phase. These observations indicate that PP2A can be targeted in a calcium-regulated manner to Cdc6 via the PR70 subunit, where it plays a role in regulating protein phosphorylation and stability.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Precise regulation of DNA replication is necessary to ensure that daughter cells receive a complete and intact genome during mitosis. A crucial step in regulating DNA replication is the assembly of pre-replicative complexes at origins of replication (1). Coordination of DNA replication with the cell cycle is achieved through a periodic accumulation and destruction of proteins involved in formation of pre-RCs3 is mediated by cyclin-dependent kinases (CDKs) and the E3 ubiquitin ligase, anaphase promoting complex/cyclosome (2). The mammalian Cdc6 protein is required for DNA replication and acts in conjunction with the Cdt1 protein to recruit the mini-chromosome maintenance complex into pre-RCs (1, 3, 4). Mammalian cells have multiple mechanisms to ensure that pre-RCs only assemble during late M and G1, including regulation of the levels and function of Cdc6 (2).

Mammalian Cdc6 is regulated by phosphorylation of multiple sites within its N-terminal domain by cyclin-dependent protein kinases. Cdc6 is phosphorylated at canonical CDK sites, including serines 54, 74, and 106 of human Cdc6 (5, 6). Experiments with exogenously expressed protein have shown that phosphorylation can regulate the nuclear localization of Cdc6 (5, 7-9). However, other studies have shown that a subpopulation of endogenous Cdc6 remains in the nucleus, bound to chromatin, throughout the cell cycle (10-12). Phosphorylation of Cdc6 also plays an important role in regulating the stability of Cdc6. The N-terminal domain of Cdc6 contains RXXL (D box) and KEN (KEN box) destruction motifs, which are binding sites for the form of the APC/C containing the cdh1-targeting subunit (13). Cdc6 is polyubiquitinated and targeted for degradation by APC/Ccdh1, which prevents formation of pre-RCs in quiescent cells and during early G1 by maintaining low levels of Cdc6 (14). Phosphorylation of Cdc6 by CDKs protects the protein from degradation by blocking recognition by cdh1 resulting in stabilization of Cdc6 during a window of time that allows formation of pre-RCs during G1 (15). The importance of CDK-mediated stabilization of Cdc6 is also supported by evidence showing that the cell cycle arrest caused by DNA damage is due to dephosphorylation and degradation of Cdc6 (16).

Because the extent of Cdc6 phosphorylation is controlled by the opposing actions of cyclin-dependent kinases and protein phosphatases, dephosphorylation of Cdc6 can also control formation of pre-RCs. Much less is known about mechanisms that regulate Cdc6 dephosphorylation. A previous study identified a fragment of PR70 as a member of the PPP2R3 family of PP2A regulatory subunits that interacted with Cdc6 and implicated PP2A in regulating Cdc6 phosphorylation (17). The major forms of PP2A contain a dimeric core complex composed of a scaffold (A) and a catalytic subunit (C). The AC core dimer associates with regulatory subunits that form heterotrimeric holoenzymes and target the catalytic subunit to specific phosphoprotein substrates (18-20). In this study, the mechanism and functional consequences of targeting of PP2A to Cdc6 by PR70 were investigated. The results show that PR70 interacts with PP2A and Cdc6 through distinct regions of the protein, that the association of PP2A to Cdc6 is enhanced by calcium binding to PR70, and that loss of PR70 causes increased levels of Cdc6 and G1 arrest.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of Full-length PR70—A human expressed sequence tag encoding the PR70 start codon was identified in the human expressed sequence tag data base using the MegaBLAST tool (www.ncbi.nlm.nih.gov/BLAST/) with the assembled PR70 sequence (21). A PR70 cDNA was constructed using the PR48 cDNA and the IMAGE Human Clone ID 5728169 (GenBankTM accession number BM544432 [GenBank] ), purchased from Invitrogen, using an internal NcoI restriction site present in the common region of BM54432 and PR48. A PCR fragment containing the translational start codon, the 5'-end, and the 3'-NcoI site of BM54432 was generated using the BM54432 cDNA as template with the PCR primers: 5'-CGGGATCCATGCCGCCCGGCAAAGT-3' (sense strand) and 5'-GCGCCTTGATCCGGC-3' (antisense strand). The PCR product was digested with the restriction enzymes BamHI and NcoI. The 3' portion of the PR48 cDNA was excised from the PR48 cDNA (17) using NcoI and HindIII, and the fragments were ligated and subcloned into the pCMV-Tag2B vector (Stratagene) digested with BamHI and HindIII. The resulting construct encoded a full-length PR70 cDNA fused to an N-terminal FLAG epitope tag. The sequence was verified by automated sequencing.

Cell Culture, Transfection, and RNA Interference—COS-7, HeLa, and U2OS cells were maintained at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in an atmosphere of 5% CO2. U2OS, obtained from the ATCC, is a human osteosarcoma cell line that expresses wild-type p53. For transient expression of proteins, cells were transfected with expression plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were harvested either 24 or 48 h after transfection. Transfection with small interfering RNA to knock down PR70 was carried out using Oligofectamine (Invitrogen) following the manufacturer's protocol. Annealed duplex siRNAs were purchased from Dharmacon and had the following sequences: PR70-1, 5'-AGCCGGUCCUGAAGAUGAAdTdT-3' (sense strand) and PR70-2, 5'-AAAGCAUUCCGACCUUCUAdTdT-3' (sense strand). Controls included an siRNA that knocks down the MEKK2 protein kinase (22), an siRNA that knocks down protein phosphatase 5 (23), and an siRNA corresponding to the sequence of firefly luciferase (5'-TCGAAGTATTCCGCGTACGdTdT-3'). Cells were lysed and analyzed by immunoblotting 48 h after transfection. In some experiments, cells were co-transfected with PR70 siRNA and expression plasmids encoding wild-type Cdc6 or Cdc6 mutants in which all three N-terminal phosphorylation sites were mutated to alanine (AAA-Cdc6) or aspartic acid (DDD-Cdc6) using Lipofectamine 2000 and harvested 48 h later. The cDNAs encoding phosphorylation site mutants of Cdc6 were prepared using a PCR-based site-directed mutagenesis kit (Invitrogen) according to the manufacturer's protocol. Wild-type and mutant Cdc6 were expressed as a fusion proteins fused to the N terminus of enhanced green fluorescent protein using the pEGFP-N expression vector (Clontech).

Immunoprecipitation and Immunoblotting—Rabbit antisera were raised against a synthetic peptide corresponding to the C terminus of human PR70 (CDLYEYACGDEDLEPL) conjugated to keyhole limpet hemocyanin. Anti-PR70 antibodies were affinity purified on a peptide column made with the same peptide using the MicroLink Peptide Coupling Kit (Pierce) following the manufacturer's protocol. Rabbit antiserum against human Cdc6 was generated against a full-length Cdc6 fusion protein as described previously (4).

Proteins were immunoprecipitated following the protocol described previously (17). Briefly, the media was aspirated and the cells were washed with cold PBS. The cells were incubated on ice for 20 min in 300 µl of IP lysis buffer containing 20 mM Tris-HCl (pH 7.5), 0.2% Nonidet P-40, 20% glycerol, 200 mM NaCl, 1 mM EDTA, and protease inhibitor mixture (Roche Applied Science). Lysates were centrifuged at 14,000 x g for 10 min, and protein complexes were immunoprecipitated from the supernatant. Endogenous PR70 and Cdc6 were immunoprecipitated from 1.2 x 106 HeLa cells lysed in 300 µl of IP lysis buffer as described above. PR70 was immunoprecipitated using a rabbit antiserum generated against the peptide CDLYEYACGDEDLEPL conjugated to hemocyanin. Cdc6 was immunoprecipitated using a rabbit polyclonal antibody generated against a full-length Cdc6-GST fusion protein described previously. As a negative control, immunoprecipitations were performed using pre-immune serum collected from the rabbits immunized against PR70 or Cdc6. 10 µl of antiserum and 40 µl of protein A-Sepharose (Sigma-Aldrich) were added to 300 µl of lysate, and the mixture was incubated for 2 h at 4 °C. The protein-A beads were washed three times with IP lysis buffer, and protein was solubilized in 60 µl of 2x SDS-PAGE loading buffer. Thirty microliters of solubilized material was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was cut into pieces, which were probed with anti-PP2A C-subunit monoclonal antibody 1F6 (24), anti-PP2A A-subunit antiserum (C-20, Santa Cruz Biotechnology), anti-Cdc6 monoclonal antibody (clone DCS-180, Upstate), or anti-PR70 antiserum. Following incubation with horseradish peroxidase-conjugated secondary antibodies, the blots were developed using the enhanced chemiluminescence detection system (Amersham Biosciences).

Transiently expressed FLAG-tagged proteins were immunoprecipitated from 1.5 x 106 cells using 7 µg of anti-FLAG polyclonal antibody (Sigma-Aldrich) or 7 µg of non-immune rabbit IgG (Sigma-Aldrich) and 40 µl of protein A-Sepharose (Sigma-Aldrich) for 2 h at 4 °C. The immunoprecipitates were washed three times with lysis buffer and solubilized in 60 µl of 2x SDS-PAGE loading buffer. 30 µl of solubilized protein was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was probed with anti-FLAG M2 monoclonal (Stratagene), anti-PP2A C-subunit 1F6, and anti-PP2A A-subunit (C-20, Santa Cruz Biotechnology) antibodies and developed as described above.

Calcium Overlay Assay—In vitro 45Ca2+ overlay assays were carried out using a protocol described previously (25). Purified GST fusion proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was washed three times in IMK buffer (10 mM imidazole-HCl, pH 6.8, 5 mM MgCl2, and 60 mM KCl) for 1 h at room temperature. The membrane was then incubated in IMK buffer containing 5 µCi/ml 45Ca2+ for 10 min. The membrane was washed three times in 30% ethanol for 5 min, dried, and exposed to x-ray film for 12 h.

Generation of PR70 Mutants—Point mutations were introduced into the EF-hands of PR70 as described in the manual for the QuikChange Multi Site-directed mutagenesis kit (Stratagene) using the following primers (mutated residues underlined). PCR was performed with pCMV-Tag2B containing the full-length PR70 cDNA as template with the following primers: EF1(x,y) 5'-CAAGTTCTGGGAGCTGGCCACGGCCCACGACCTGCTCATCG-3' (sense strand) and 5'-CGATGAGCAGGTCGTGGGCCGTGGCCAGCTCCCAGAACTTG-3' (antisense strand), EF1(-z) 5'-TTGTGCCGCGCCAGGTTGTCCGCGTCGATGAGCGCTCATCGACGCGGACAACCTGGCGCGGCACAA-3' (sense strand) and 5' -> 3' (antisense strand), EF2(x,y) 5'-TGGTTCCGCTGCATGGCCCTGGCCGGGGACGGCGCCCTG-3' (sense strand) and 5'-CAGGGCGCCGTCCCCGGCCAGGGCCATGCAGCGGAACCA-3' (antisense strand), and EF2(-z), 5'-GCGCCCTGTCCATGTTCCAGCTCGAGTACTTCTAC-3' (sense strand) and 5'-GTAGAAGTACTCGAGCTGGAACATGGACAGGGCGC-3' (antisense strand). Mutations in both EF-hands were introduced using the EF1 mutant cDNAs as template for PCR with primers for introduction of EF2 point mutants. All mutations were verified by automated sequencing.

PR70 truncation mutants were generated by PCR amplification using the PR70 cDNA as template. The {Delta}N1 (aa 125-575) corresponds to the PR48 protein described previously (17). {Delta}N2, {Delta}N3, and {Delta}C were generated using the following primers: {Delta}N2 (aa 136-575) 5'-CGGGATCCGCCACCATGGATGACATG-3', {Delta}N3 (aa 162-575) 5'-CGGGATCCAGGACTCCGTCAACGTG-3', and {Delta}C (aa 1-441) 5'-CCCAAGCTTCATCTGGCAGAGGCAGTC-3'.

Point mutations were introduced into the FYF motif (aa 128-130) of PR70 using the full-length PR70 cDNA as template with the following mutagenic primers (mutated residues underlined): AYF, 5'-GCCAAAGCATTCCGACCGCCTACTTCCCCAGAGGACG-3' (sense strand) and 5'-CGTCCTCTGGGGAAGTAGGCGGTCGGAATGCTTTGGC-3' (antisense strand), FAF, 5'-CCAAAGCATTCCGACCTTCGCCTTCCCCAGAGGACGCC-3' (sense strand) and 5'-GGCGTCCTCTGGGGAAGGCGAAGGTCGGAATGCTTTGG-3' (antisense strand), FYA, 5'-GCATTCCGACCTTCTACGCCCCCAGAGGACGCCCGC-3' (sense strand) and 5'-GCTTTCGTCCTCTGGGGGCGTAGAAGGTCGGAATGC-3' (antisense strand). To make the AYAP mutant, the AYFP cDNA was used as a template, and PCR mutagenesis was done with the following primers: AYA, 5'-CATTCCGACCGCCTACGCCCCCAGAGGACGCCCG-3' (sense strand) and 5'-CGGGCGTCCTCTGGGGGCGTAGGCGGTCGGAATG-3' (antisense strand). To make the AAAP mutant, the AYAP cDNA was used as a template, and PCR mutagenesis was done with the following primers AAA, 5'-GCATTCCGACCGCCGCCGCCCCCAGAGGACG-3' (sense strand) and 5'-CGTCCTCTGGGGGCGGCGGCGGTCGGAATGC-3' (antisense strand). All mutations were verified by automated sequencing.

Cloning of GST-tagged PR70 and EF-hand Mutants—PR70 and PR70 EF-hand mutant cDNA were cloned into the pGEX-4T-1 vector (Amersham Biosciences). The cDNAs were amplified by PCR using the pCMV-Tag2B vector containing the full-length PR70 or EF-hand mutant cDNA as template with the following primers: 5'-CGGGATCCATGCCGCCCGGCAAAGT-3 (sense strand) and 5'-ATTTGCGGCCGCTCACAGCGGCTCCAGGTC-3' (antisense strand). The products were digested with BamHI and NotI and ligated into pGEX 4T-1, which had been cut with the same restriction enzymes. The resulting constructs encode the GST protein fused to the N terminus of full-length PR70 or EF-hand mutant proteins. The sequences were verified by automated sequencing.

Expression and Purification of GST-Cdc6, GST-A, and GST-PR70 Fusion Proteins—A GST-Cdc6 fusion protein was prepared by a modification of a method previously described (6). Briefly, 1 liter of Sf9 cells (2 x 106 cells/ml) was infected with recombinant GST-Cdc6 baculovirus (a gift of Dr. Ellen Fanning, Vanderbilt University) at a Sf9 culture:baculovirus ratio of 1:20 (v/v) for 60 h. The cells were collected by centrifugation and washed once with PBS. Cells were lysed on ice in 40 ml of buffer A (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM dithiothreitol, 10 mM NaF, 1 mM EGTA, 2 mM EDTA, and a protease inhibitor tablet (Roche Applied Science)) using a Dounce homogenizer. Lysates were centrifuged at 30,000 x g for 30 min at 4 °C to remove cellular debris, and the lysate was mixed with 2 ml of glutathione-agarose (Sigma-Aldrich) for 2 h at 4 °C. The resin was recovered by centrifugation and washed twice with PBS, once with PBS containing 1.5 M NaCl, and once with PBS containing 1.5 mM NaCl and 0.1% (v/v) Nonidet P-40, and then re-equilibrated in PBS. The GST-Cdc6 fusion protein immobilized on glutathione agarose beads was resuspended in buffer B (20 mM HEPES, pH 7.6, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, and 50% glycerol) and stored at -80 °C.

GST-A fusion protein, GST-PR70, and GST-EF-hand mutants were expressed in bacteria and prepared as previously described (26). The GST fusion proteins immobilized on glutathione agarose beads were resuspended in buffer B and stored at -80 °C until use.

GST Pulldown Assays—GST, GST-A, and GST-Cdc6 immobilized on glutathione-agarose beads were used to assess the binding of PR70. Wild-type or mutant FLAG-PR70 was expressed by transient transfection of COS-7 cells. The cells were lysed on ice in 300 µl of IP lysis buffer or in 300 µl of IP lysis buffer containing 10 mM EDTA or 10 mM CaCl2 for 20 min. GST pulldown assays (26) were conducted by incubating 300 µl of lysate with either GST, GST-A, or GST-Cdc6. The samples were incubated for 1 h at room temperature with agitation. The calpain inhibitor calpeptin (Calbiochem) was added at a concentration of 50 µM in some experiments. Following incubation, the sample was washed three times with IP lysis buffer supplemented with EGTA, CaCl2, or CaCl2 and calpeptin, and the beads were collected by centrifugation. After washing, the bound proteins were solubilized in 60 µl of 2x SDS-PAGE loading buffer. 30 µl of solubilized protein was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was probed with anti-FLAG monoclonal (M2, Stratagene), anti-PP2A C-subunit 1F6, and anti-PP2A A-subunit (C-20, Santa Cruz Biotechnology) antibodies and developed as described above.


Figure 1
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  		FIGURE 1.
PR70 interacts with Cdc6 and PP2A. A, Cdc6 was immunoprecipitated from exponentially growing HeLa cells using a polyclonal antiserum specific for Cdc6 (Cdc6) or pre-immune serum (Pre). The immunoprecipitates and supernatant fractions were analyzed by immunoblotting using anti-Cdc6 (Cdc6), anti-PR70, anti-A-subunit, or anti-C-subunit antibodies. B, PR70 was immunoprecipitated from HeLa cells using an anti-peptide antiserum against PR70 (PR70) or pre-immune serum (Pre). The immunoprecipitates (IP) or the supernatants remaining after immunoprecipitation (S) were resolved by SDS-PAGE and analyzed by immunoblotting with anti-PR70 (PR70), anti-A-subunit (A), and anti-C-subunit (C) antibodies as indicated on the left. C, HeLa cells were transiently transfected with empty expression vector (Emp Vec), plasmids expressing the FLAG-PR70{Delta}N mutant ({Delta}N1), full-length FLAG-PR70, or a combination of plasmids expressing FLAG-PR70 and HA-Cdc6 (Cdc6). Cells were harvested after 24 h, and lysates were immunoprecipitated with anti-FLAG antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-FLAG (FLAG-PR70), anti-Cdc6, anti-A-subunit, and anti-C-subunit antibodies as indicated on the left.

 
Flow Cytometry—U2OS cells (4 x 105) were seeded into 60-mm dishes and transfected with PR70 or control siRNA 24 h later. The cells were then incubated for 48 h and harvested by trypsinization, washed once with PBS, and resuspended in 0.5 ml of PBS. The cell suspension was then added to 4.5 ml of 70% ethanol and incubated on ice for 2 h. Cells were collected by centrifugation, washed once with PBS, and suspended in 1 ml of propidium iodide/Triton X-100 staining solution with RNase (0.1% Triton X-100, 0.2 mg/ml DNase-free RNase, and 10 µg/ml propidium iodide in PBS). The DNA content of 10,000 cells was determined using a BD Biosciences FACScan flow cytometer and FlowJo software. Single cells were gated away from clumped cells using an FL3 width versus FL3 height dot plot, and the DNA content of individual cells was plotted as FL3 area versus cell number.

Experimental Reproducibility—The data shown in the figures are from individual experiments that were representative of common results obtained in at least three independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Interaction of PR70 with Cdc6—The original cDNA for PR70, termed PR48, was identified in a yeast two-hybrid screen using the human Cdc6 protein as bait (17) and subsequently shown to be a fragment of a longer cDNA (27). A full-length human PR70 cDNA was constructed by ligating expressed sequence tag BM54432 to the PR48 cDNA using an internal NcoI restriction site. The predicted open reading frame of the PR70 cDNA encodes a protein with a calculated molecular mass of 65.1-kDa and corresponds to the longer transcript (variant 1) of the PPP2R3B gene (GeneID: 28227). The predicted amino acid sequence of PR70 is highly similar to the human PR72 and mouse PR59 members of the PPP2R3 gene family, but more distantly related to the G5PR protein (supplemental Table S1). An alignment of the PPP2R3 family (supplemental Fig. S1) revealed a highly conserved central domain, termed the R3 domain, that contains two conserved EF-hand calcium binding motifs previously identified in PR72 (27). Rabbit antisera were raised against a peptide corresponding to the unique C terminus of PR70 and affinity purified on a peptide column. The purified antibodies recognized a protein band of Mr = 70,000 in lysates of HeLa cells. The 70-kDa protein recognized by the antibody was greatly reduced in cells treated with two different siRNAs corresponding to sequences within PR70 but not with control siRNA (supplemental Fig. S2).

To verify that PR70 associates with PP2A and Cdc6, HeLa lysates were immunoprecipitated with PR70 and Cdc6 antibodies. Immunoprecipitation of Cdc6 co-precipitated a diffuse protein band that migrated at the position of PR70 that was not present in immunoprecipitates obtained with the preimmune serum (Fig. 1A). The anti-Cdc6 serum also co-precipitated the A- and C-subunits of PP2A. Although the anti-PR70 antibodies co-precipitated the A- and C-subunits of PP2A (Fig. 1B), a complex between PR70 and Cdc6 could not be detected. The inability to detect Cdc6 may be due to steric hindrance by the anti-C-terminal antibody, because the C terminus of PR70 is required for interaction with Cdc6 (see below). The association of PR70 with PP2A and Cdc6 was also tested in co-immunoprecipitation experiments using exogenously expressed proteins. FLAG-tagged PR70 was expressed in HeLa cells and immunoprecipitated with anti-FLAG antibodies. Analysis of the immunoprecipitates by immunoblotting showed that the endogenous A- and C-subunits of PP2A and HA-tagged Cdc6 co-precipitated with FLAG-PR70 (Fig. 1C). These results indicate that PR70 can interact with both the PP2A core dimer and Cdc6 in intact cells.


Figure 2
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  		FIGURE 2.
Interaction of PR70 with PP2A is enhanced by calcium-binding. A, the figure shows a diagram of the location and sequences of wild-type PR70 (WT) and PR70 mutants containing substitutions of calcium-binding residues within the EF-hand motifs (EF mutants). The canonical EF-hand residues involved in coordination of calcium are indicated by the letters x, y, z, -x, -y, and -z using standard nomenclature (28). The x, y, and -z residues that were mutated are shown in bold type. B, GST fusions of wild-type PR70 (PR70) and the EF-hand mutant were analyzed for calcium binding by 45Ca2+ overlay assay. The amounts of GST-PR70 in each lane were determined by staining the gel with Coomassie Brilliant Blue (CBB). C, calcium enhances binding of PR70 to the A-subunit of PP2A but not to Cdc6. FLAG-PR70 and the indicated EF-hand mutants were transiently expressed in COS-7 cells, and the cells were lysed in standard buffer (N) or lysis buffer containing EGTA (E) or CaCl2 (Ca). Calpeptin (50 µM) was included in some experiments (Ca+CP). The lysates were incubated with GST alone (GST), GST-A, or GST-Cdc6, and bound proteins were detected by immunoblotting with anti-FLAG (FLAG-PR70), anti-A-subunit (A), anti-C-subunit (C), and anti-GST (GST) antibodies.

 
Calcium Enhances the Interaction of PR70 with the AC Core Dimer and Recruits PP2A to Cdc6—Analysis of the amino acid sequence of PR70 identified two EF-hand calcium binding motifs that are conserved within the PPP2R3 family (supplemental Fig. S1). The roles of these motifs were tested in a gel overlay assay with wild-type PR70 and PR70 containing inactivating mutations of the EF-hand motifs. Point mutants were constructed that had substitutions of amino acids involved in calcium binding (28), including alanine substitutions at both the x and y coordinates and a conservative change at the -z coordinate (Fig. 2A). Wild-type PR70 bound calcium in the in vitro 45Ca2+ overlay assay (Fig. 2B). Mutation of the first EF-hand (EF1) resulted in reduced binding of calcium compared with wild-type PR70. Mutation of the second EF-hand (EF2) severely reduced calcium binding, whereas the double mutation of EF1 and EF2 nearly abolished the ability of PR70 to bind calcium.

Calcium binding causes a conformational change in the PR72 member of the PPP2R3 family that is associated with enhanced interaction with the A-subunit of PP2A (27). Therefore, the effects of calcium on the interaction of PR70 with PP2A and Cdc6 were determined. FLAG-tagged PR70 was expressed in COS-7 cells, which were lysed in buffer containing EGTA or calcium. The lysates were incubated with either GST-A or GST-Cdc6 and bound proteins detected by immunoblotting. FLAG-PR70 interacted with both GST-A and GST-Cdc6 but not GST alone (Fig. 2C, lanes 1-2 and 6-7). Compared with lysates prepared with standard buffer or EGTA, the addition of calcium enhanced the binding of PR70 to GST-A, but not to GST-Cdc6 (Fig. 2C, lanes 4 and 9). Although calcium did not enhance the binding of PR70 to GST-Cdc6, it did cause a significant increase in the amount of A- and C-subunits associated with GST-Cdc6 (Fig. 2C, lanes 9 and 10). Although an excess of calcium was used in the experiments shown in Fig. 2C, other experiments showed that enhanced binding of the AC core dimer was also observed at calcium concentrations of 100 µM (not shown).

To test the function of the individual EF-hand motifs in the calcium-enhanced interaction with PP2A, GST pulldown experiments were performed with the calcium-binding mutants. Compared with assays in the presence of EGTA, the addition of calcium resulted in enhanced binding of wild-type PR70 and the EF1 mutant to GST-A, but not to GST-Cdc6 (Fig. 3A and B, lanes 2-5). Although it did not increase the amount of PR70 or the EF1 mutant associated with Cdc6, calcium did increase the association of the A- and C-subunits with GST-Cdc6 (Fig. 3B, lanes 2-5). Mutation of EF2 or mutation of both EF1 and EF2 resulted in loss of the calcium-enhanced binding of PR70 to GST-A (Fig. 3A, lanes 6-9) and the calcium-dependent association of the A- and C-subunits with GST-Cdc6 (Fig. 3B, lanes 6-9).

The effect of calcium on the interaction of PR70 with PP2A was also assessed by expression and immunoprecipitation in COS-7 cells. Both wild-type PR70 and the EF1 mutants interacted with endogenous PP2A (Fig. 3C, lanes 2-4). The EF1(-z) mutant interacted as well as wild-type PR70, but interaction of the EF1(x,y) mutant was reduced suggesting that mutation of the x and y residues causes a structural defect in PR70. Mutation of EF2, or both EF1 and EF2, resulted in a nearly complete loss of interaction with PP2A (Fig. 3C, lanes 5-8). A longer exposure of the blot showed that a weak interaction of the A- and C-subunits with the EF2 and EF1/EF2 double mutants could still be detected (Fig. 3C, OE). The combination of intact cell data and in vitro binding assays provide evidence that PR70 is a calcium binding protein and that interaction with the core dimer of PP2A is enhanced by binding of calcium to the second EF-hand motif. Calcium does not affect interaction of PR70 with Cdc6 but increases the association of the PP2A core dimer with Cdc6 in a manner dependent upon binding of calcium to the second EF-hand of PR70.


Figure 3
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  		FIGURE 3.
The calcium-enhanced association of PP2A with PR70 requires EF2. FLAG-PR70 and the indicated EF-hand mutants were transiently expressed in COS-7 cells, and the cells were lysed in standard lysis buffer (N) or lysis buffer containing EGTA (E) or CaCl2 (Ca). A, GST pulldown assays were performed with the different lysates using immobilized GST-A. Bound proteins were detected by immunoblotting with anti-FLAG (FL-PR70), anti-A-subunit (A), and anti-C-subunit (C) antibodies. B, GST pulldown assays were performed using immobilized GST-Cdc6 as described for A. Lane 1 of panels A and B shows a control pulldown assay using GST alone. C, COS-7 cells were transiently transfected with FLAG-tagged wild-type PR70 or the indicated EF-hand mutants, and lysates prepared with standard buffer were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted as described in A. Overexposures of the anti-A-subunit (AOE) and anti-C-subunit (COE) immunoblots are also shown.

 


Figure 4
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  		FIGURE 4.
Mapping of PP2A binding domains in PR70. A, a schematic diagram of PR70 showing the region containing the conserved R3 domain and PR70-unique regions. The truncation mutants used in binding assays are shown below with their corresponding designations on the left and amino acid numbers on the right. The conserved FYF (FYF motif) and EF-hand motifs (EF1 and EF2) are also depicted. B, interaction of PR70 truncation mutants with the A- and C-subunits of PP2A. FLAG-tagged PR70 (PR70) and the indicated truncation mutants were transiently expressed in COS-7 cells. The cells were lysed and the FLAG-tagged proteins immunoprecipitated with anti-FLAG antibody (Anti-FLAG IP). The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-FLAG (FLAG), anti-A-subunit (A), and anti-C-subunit (C) antibodies. A control immunoprecipitate using lysate from cells transfected with the empty expression vector (Emp Vec) is shown in the first lane.

 
PP2A and Cdc6 Bind to Distinct Regions of PR70—Comparison of the amino acid sequences of the PPP2R3 regulatory subunits identified a conserved domain in the central region of PR70 (supplemental Figs. S1 and S4A). The R3 domain is 66% identical and 82% conserved between human PR70 (PPP2R3B) and PR72 (PPP2R3A). A series of truncation mutants were constructed to identify regions within PR70 that were important for interaction with PP2A and Cdc6. FLAG-tagged mutants were expressed in COS-7 cells and immunoprecipitated with anti-FLAG antibody. The ability of the mutants to incorporate into endogenous PP2A heterotrimers was determined by immunoblotting for associated A- and C-subunits. The {Delta}N1 mutant contains a deletion of the entire N-terminal PR70-unique region and interacted with endogenous PP2A subunits to the same extent as full-length PR70 (Fig. 4B). Deletion of a C-terminal segment that included the PR70-unique region ({Delta}C) had little, if any, effect on the interaction with PP2A. However, deletions of N-terminal regions of the conserved R3 domain, {Delta}N2 and {Delta}N3, resulted in proteins that failed to interact with PP2A. These data indicated that the region between amino acids 125 and 136 of PR70 were necessary for interaction with the PP2A core dimer.

The sequence between residues 125 and 136 of PR70 contains a hydrophobic motif (FYF) that was conserved in PPP2R3 proteins from humans to flies (Fig. 5A). The role of the FYF motif was tested by mutating these residues to alanines (Fig. 5B) and determining the effects on interaction with the AC core dimer. Mutation of any one of these residues resulted in a significant loss of interaction with endogenous PP2A (Fig. 5C). A longer exposure of the immunoblot showed that small amounts of the A- and C-subunits could be detected in immunoprecipitates of each of the mutants (not shown). Although the interaction of the FYF mutants was severely compromised in intact cells, these mutants still bound to PP2A when assayed in vitro by GST pulldown experiments (not shown). These results indicate that the FYF motif contributes to the interaction of PR70 with the A-subunit.

The N- and C-terminal truncation mutants of PR70 were also tested for their ability to interact with Cdc6. Pulldown experiments with GST-Cdc6 were performed with full-length PR70, the {Delta}N3, and the {Delta}C mutants in the presence and absence of calcium. As expected, full-length PR70 and the {Delta}C mutant interacted with the A-subunit of PP2A, whereas the {Delta}N3 mutant did not (Fig. 6A). Furthermore, there was an enhanced interaction of PR70 and the {Delta}C mutant in the presence of calcium. As shown previously with full-length PR70 (Fig. 2C), the enhanced binding of PR70 and the {Delta}C mutant was accompanied by a decrease in the amount of C-subunit associated with GST-A (Fig. 6A, lanes 3 and 9). This decrease in associated C-subunit was not observed with the {Delta}N3 mutant, which did not bind to GST-A. These observations suggest that the decrease in C-subunit in the presence of calcium is due to displacement of endogenous regulatory and catalytic subunits from GST-A by excess free PR70.


Figure 5
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  		FIGURE 5.
A conserved hydrophobic motif is involved in the interaction of PR70 with PP2A. A, an alignment of the N-terminal region of the conserved R3 domains (residues 125-135 of PR70) of PPP2R3 subunits from various species. Conserved FYF residues are shown in bold. B, a diagram showing the residues within the PR70 that were changed in the mutant forms of PR70 listed on the left. Amino acid substitutions are shown in bold. C, FLAG-tagged PR70 (PR70), the {Delta}N3 mutant ({Delta}N3), and the indicated FYF mutants were transiently expressed in COS-7 cells. The cells were lysed, and tagged proteins were immunoprecipitated with anti-FLAG antibody (Anti-FLAG IP). The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-FLAG (FLAG), anti-A-subunit (A), and anti-C-subunit (C) antibodies. A control immunoprecipitate using lysate from cells transfected with the empty expression vector (Emp Vec) is shown in the first lane.

 


Figure 6
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  		FIGURE 6.
The C-terminal region of PR70 mediates interaction with Cdc6. FLAG-PR70 (lanes 1-3), the {Delta}N3 mutant (lanes 4-6), and the {Delta}C mutant (lanes 7-9) were transiently expressed in COS-7 cells, and the cells were lysed in the presence of EGTA (E) or CaCl2 (Ca). GST pulldown assays were performed using immobilized GST-A (panel A) or GST-Cdc6 (panel B), and bound proteins were detected by immunoblotting with anti-FLAG (FLAG), anti-A-subunit (A) or anti-C-subunit (C) antibodies. Control pulldowns with GST alone (GST) were carried out with all three expressed proteins using lysates prepared with standard buffer (lanes 1, 4, and 7).

 
Both full-length PR70 and the {Delta}N3 mutant bound to GST-Cdc6 (Fig. 6B). However, only full-length PR70 was able to recruit additional A- and C-subunits in the presence of calcium. In contrast, the {Delta}C mutant bound very poorly to Cdc6 in the presence or absence of calcium. A low level of the A- and C-subunits was pulled down with GST-Cdc6 from lysates expressing the {Delta}N3 or {Delta}C mutants (Fig. 6B, lanes 5-6 and 8-9). Similar amounts of these subunits were also bound to GST-Cdc6 in lysates from non-transfected cells (not shown) suggesting that GST-Cdc6 can interact with endogenous A- and C-subunits in the absence of expressed PR70 (presumably by binding to endogenous PR70). The amounts of A- and C-subunits bound to GST-Cdc6 in experiments with the {Delta}N3 or {Delta}C mutants were increased in the presence of calcium. This observation provides additional support for the conclusion that calcium can regulate the association of PP2A with Cdc6 and shows that a C-terminal region of PR70, which includes the PR70-unique domain, is necessary for interaction with Cdc6.

PR70 Regulates Cdc6 Levels—Because phosphorylation of the N-terminal regulatory sites of Cdc6 inhibits degradation, loss of the phosphatase that dephosphorylates these sites should promote accumulation of Cdc6. Therefore, RNA interference was used to determine if knockdown of PR70 affected the levels of Cdc6. Knocking down the catalytic subunit of PP2A increased the levels of endogenous Cdc6 in HeLa cells (Fig. 7A). Treatment of cells with either a control siRNA or an siRNA that knocks down protein phosphatase 5 had no effect on Cdc6 levels. Knocking down the PR70 subunit also caused a substantial increase in the levels of Cdc6 (Fig. 7B). The increase in Cdc6 levels occurred with two PR70 siRNAs targeted to distinct regions of the mRNA. The accumulation of Cdc6 following knockdown with PR70-1 siRNA appeared to be greater than that with PR70-2 siRNA, which is consistent with the greater efficiency of the PR70-1 siRNA in reducing PR70 levels (supplemental Fig. S2). Phosphorylation site mutants of Cdc6 were then used to test the role of phosphorylation in the accumulation of Cdc6 caused by knockdown of PR70. Knockdown of PR70 caused an increase in the levels of expressed wild-type GFP-Cdc6 compared with transfection with a control siRNA (Fig. 7B). Transfection with a mutant of Cdc6 in which all three N-terminal phosphorylation sites had been mutated to phospho-mimicking aspartic acid residues (DDD-Cdc6) resulted in substantially higher levels of expression than those observed with the wild-type protein as previously reported (15). PR70-1 siRNA had little or no effect on the levels of DDD-Cdc6. The ability of PR70 knockdown to cause accumulation of Cdc6 was also greatly diminished when the phosphorylation sites were mutated to non-phosphorylatable alanine residues (AAA-Cdc6). Similar results were seen in U2OS cells. These data indicate that knockdown of PR70 results in an increase in the levels of endogenous and exogenous Cdc6 that is dependent on the presence of phosphorylatable residues at the N-terminal phosphorylation sites.


Figure 7
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  		FIGURE 7.
Knockdown of PR70 increases the levels of Cdc6. A, HeLa cells were mock transfected (Mock) or transfected with control (Luc), PP2A catalytic subunit (PP2A-C), or PP5 (PP5) siRNA. Cells were harvested 48 h after transfection and immunoblotted with anti-Cdc6, PP2A-C, PR70, or PP5 antibodies. B, HeLa cells were mock transfected (Mock) or transfected with control (Luc), PR70-1, or PR70-2 siRNA. Cells were harvested 48 h after transfection and immunoblotted with anti-Cdc6, PR70, or actin (as a loading control) antibodies. C, HeLa cells were co-transfected with control or PR70-1 siRNA and plasmids encoding GFP-tagged versions of wild type Cdc6 (wtCdc6) or Cdc6 in which the N-terminal phosphorylation sites were mutated to aspartic acid (DDD-Cdc6) or alanine (AAA-Cdc6). Forty-eight hours later, the cells were harvested and lysates were analyzed by immunoblotting with antibodies against Cdc6 or glyceraldehyde-3 phosphate dehydrogenase (GPDH) as a loading control. D, duplicate samples of the lysates described in B, from cells co-transfected with Cdc6 and either luciferase control (Luc siRNA) or PR70-1 (PR70) siRNAs were immunoblotted with anti-PR70 antibodies to confirm knockdown of PR70.

 


Figure 8
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  		FIGURE 8.
Overexpression of PR70 increases the levels of Cdc6. A, U2OS cells were transfected with empty vector, or plasmids encoding GFP-Cdc6 (Cdc6), myc-tagged CDK2 (CDK2), or FLAG-tagged constructs of wild-type PR70 (PR70) or the indicated PR70 mutants. Cells were harvested 24 h after transfection, and lysates were analyzed by immunoblotting with antibodies against Cdc6, actin, FLAG, or myc as indicated at the right. The Cdc6 antibodies detected both the expressed Cdc6 (GFP-Cdc6) and endogenous Cdc6 (Endo-Cdc6). B, U2OS cells were co-transfected with empty vector or plasmids expressing FLAG-PR70 and plasmids expressing GFP-tagged versions of wild-type Cdc6 (wtCdc6), or the AAA (AAA-Cdc6), or DDD (DDD-Cdc6) triple phosphorylation site mutants of Cdc6. Cells were harvested 24 h later, and lysates were analyzed by immunoblotting with antibodies against Cdc6, actin, or the FLAG epitope as indicated at the right.

 
The effects of overexpressing PR70 on Cdc6 levels were also determined. When HeLa cells were transfected with expression plasmids containing the CMV promoter, FLAG-tagged PR70 was expressed at levels 5- to 10-fold higher than the endogenous protein (not shown). Co-expression of CDK2 and Cdc6 caused a substantial increase in Cdc6 levels as reported previously (15). Expression of wild-type PR70 caused an increase in the levels of both co-transfected and endogenous Cdc6 (Fig. 8A). Expression of the {Delta}N3 or EF1/2 mutants, which cannot interact with the AC core dimer but bind to Cdc6, also increased the levels of Cdc6. In contrast, expression of the {Delta}C mutant, which binds to the AC core dimer but not to Cdc6, had relatively little effect on the levels of exogenous or endogenous Cdc6. The potential role of phosphorylation in the effects of overexpressed PR70 was tested using the phosphorylation site mutants of Cdc6. Although co-expression of PR70 caused some increase in the levels of the AAA mutant of Cdc6, the effect was much less than its effect on wild-type Cdc6 (Fig. 8B). Similarly, co-expression of PR70 had little effect on the levels of the DDD mutant of Cdc6 even though endogenous Cdc6 was increased. Thus, the ability of overexpressed PR70 to cause accumulation of the protein was inhibited when the phosphorylation sites in Cdc6 were mutated. The effects of PR70 overexpression to cause accumulation of Cdc6 suggest it acts in a dominant-negative manner to block Cdc6 dephosphorylation (see "Discussion").


Figure 9
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  		FIGURE 9.
Knockdown of PR70 causes G1 arrest. A-D, U2OS cells were left untreated (A), or transfected with control siRNA (B), PR70-2 siRNA (C), or PR70-1 siRNA (D). Forty-eight hours later, the cells were harvested and analyzed by flow cytometry. The data are plotted as the number of cells versus DNA content determined by FL3 area. The percentages of cells in G0/G1, S, and G2/M phases are indicated. E, duplicate transfections were harvested after 48 h and immunoblotted with anti-PR70 antibody to confirm knockdown.

 
Knockdown of PR70 Causes G1 Arrest—The potential role of PR70 in progression through G1 was determined by determining the cell cycle distribution of cells in which PR70 was depleted by RNA interference. Knockdown of PR70 caused accumulation of cells in G0/G1 and depletion of cells in S and G2/M (Fig. 9). The apparent G1 arrest occurred with either of two siRNAs that target distinct regions of PR70. The level of G1 arrest correlated with the extent of PR70 knockdown. The lower levels of PR70 achieved with the PR70 siRNA-1 compared with PR70 siRNA-2 corresponded to a greater increase in the number of G1 cells (76% versus 65%). The G1 arrest caused by knockdown of PR70 supports a role for this PP2A regulatory subunit in progression through G1 phase.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The formation of pre-replicative complexes during the initiation of DNA replication is regulated, in part, by the availability of Cdc6. Cyclin-dependent kinases phosphorylate regulatory sites within the N-terminal domain of Cdc6 and block ubiquitination by APC/Ccdh1 and subsequent degradation by the proteasome (15). The results reported here help establish the form of PP2A complexed with the PR70 regulatory subunit as a physiological Cdc6 phosphatase and are consistent with a model in which PR70 targets PP2A to Cdc6 through direct protein-protein interactions. Knockdown of PR70 by RNA interference results in an increase in the levels of Cdc6 protein, consistent with a role for this subunit in regulating the stability of Cdc6. Overexpression of PR70 appeared to act in a dominant-negative manner to also increase the levels of Cdc6. The observations that increased protein levels did not occur with phosphorylation site mutants of Cdc6 are consistent with a role for PR70 in regulating Cdc6 phosphorylation and stability. A novel aspect of this model is the potential regulation of Cdc6 dephosphorylation by calcium. Calcium enhances the recruitment of the core dimer of PP2A to Cdc6 by binding to the EF-hand motifs of PR70, raising the possibility that changes in intracellular calcium can regulate the accumulation of Cdc6 and initiation of DNA replication. However, it remains to be determined if physiological changes in intracellular calcium concentrations are sufficient to regulate association of PP2A with Cdc6.

Biochemical analysis of the interaction of PR70 with the AC core dimer suggested an unanticipated mechanism for regulating PP2A activity. Experiments with N-terminal truncation mutants showed that PR70 can associate with Cdc6 independently of the A- and C-subunits. This observation contrasts with the prevailing view of PP2A in which the regulatory subunits have been thought to be constitutively associated with the core dimer (18-20). The importance of the existence of PP2A in heterotrimeric forms is supported by data showing that some PP2A regulatory subunits are only stable when incorporated into holoenzymes (29-31). Overexpression of the PPP2R2 family member, B{gamma}, leads to proteasome-dependent degradation of the free protein but not protein incorporation into holoenzymes (31). In contrast, several lines of evidence indicate that members of the PPP2R3/PR72 family are stable regardless of whether or not they are incorporated into holoenzymes. As shown here, mutants of PR70 that cannot bind to PP2A accumulate to the same levels as the wild-type protein. The apparent stability of PR70 is also independent of interaction with Cdc6, because a mutant ({Delta}C) that interacts poorly with Cdc6 accumulates to similar levels. Similarly, mutations of the related PR72 subunit that block interaction with the AC core dimer have no effect on the levels of expressed protein (27). In addition, the PR59 subunit is not degraded following loss of the A-subunit (31). Thus, in contrast to the PPP2R2/B and PPP2R5/B56 families of PP2A regulatory subunits, members of the PPP2R3/PR72 family are stable proteins whose levels and functional interactions with substrates and other proteins may be independent of the core dimer of PP2A.

The stability of expressed PR70 may also account for its ability to act in an apparent dominant-negative manner to increase the levels of Cdc6. Excess free PR70 would associate with Cdc6 and displace endogenous PR70-AC holoenzyme. Loss of the active AC core dimer from Cdc6 would inhibit dephosphorylation leading to decreased ubiquitination by APC/Ccdh1 and increase protein levels. A dominant-negative action of overexpressed PR70 is supported by observations that the effects on Cdc6 levels are not dependent on interaction of PR70 with the AC core dimer (e.g. the {Delta}N3 and EF1/2 mutants) but are dependent on interaction with Cdc6 (e.g. the {Delta}C mutant). Like knockdown of PR70, the dominant-negative actions of PR70 to increase Cdc6 levels appear to be dependent on intact phosphorylation sites, because the levels of co-expressed DDD and AAA mutants of Cdc6 were not significantly affected. Forced overexpression of the related PR72 subunit has also been reported to act in a dominant-negative manner. Expression of either wild-type PR72 or an EF-hand 2 mutant, which cannot bind the AC core dimer, both cause G1 arrest in U2OS cells (27).

The accumulation of cells with G0/G1 DNA content following knockdown by RNA interference is consistent with an important role for PR70 in progression of cells through into S phase. Similarly, overexpression of a fragment that contains the complete R3 domain and the C terminus of PR70 (termed PR48 or {Delta}N1 in this study) also causes G1 arrest, presumably through a dominant-negative action (17). The G1 arrest in cells depleted of PR70 coincides with an increase in Cdc6 protein levels. A previous study showed that increases in phosphorylation and stability of Cdc6 enhance formation of pre-replicative complexes (15). The increase in pre-replicative complex formation would be expected to enhance entry into S phase. Consistent with this idea, expression of exogenous wild-type Cdc6 leads to accelerated entry into S phase (14). However, exogenous expression of a non-phosphorylatable (AAA) mutant of Cdc6 (5) or an N-terminally truncated version of Cdc6, missing the CDK phosphorylation sites and destruction motifs recognized by APC/Ccdh1 (14), inhibit initiation of DNA replication and entry into S phase. It is possible that, even though phosphorylation is required for stabilization of Cdc6 and assembly of pre-replicative complexes during G1, an additional Cdc6 dephosphorylation or degradation step is needed to initiate DNA replication. Knockdown or overexpression of PR70 might inhibit this step and retard entry into S phase. Although the ability of PR70 knockdown to cause G1 arrest is consistent with regulation of Cdc6, an equally likely possibility is that PR70 plays other roles during G1. PR70 may regulate the activity of other proteins involved in cell cycle control, either through PP2A-mediated dephosphorylation or actions that are independent of PP2A.

The effects of calcium on the PR70-dependent association of PP2A with Cdc6 are consistent with a more general role for the PPP2R3/PR72 family in mediating calcium-regulated dephosphorylation. All four members of this family contain conserved EF-hand sequence motifs (supplemental Fig. S1). The EF-hands of PR72 are also functional calcium binding sites, and, similar to PR70, calcium binding to the second EF-hand enhances interaction with the A-subunit (27). PR72 has been shown to mediate calcium-dependent dephosphorylation of threonine-75 in the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). This report showed that, in addition to the role of EF-hand 2 in interaction with the AC core dimer, calcium binding to EF-hand 1 increased the phosphatase activity of the PR72-holoenzyme toward DARPP-32 (32). Both PR72 and its alternative splice variant (PR130) have been reported to interact with the mammalian Naked cuticle protein and regulate Wnt signaling (33, 34). Calcium may therefore influence Wnt signaling through recruitment and/or regulation of PP2A associated with Naked cuticle. The other member of the PPP2R3 family, PR59, targets PP2A to the retinoblastoma-related p107 protein (35) and may provide a mechanism for calcium regulation of the cell cycle functions of p107.

The sites involved in the interaction with PP2A and Cdc6 mapped to distinct regions of PR70, consistent with a role in bridging the two proteins. The N-terminal domain of PR70, which is not conserved with other members of the PPP2R3 family, is not required for interaction with either PP2A or Cdc6. Deletion of the C-terminal region, including the PR70-unique sequence and a portion of the conserved R3 domain, had no effect on interaction with the PP2A core dimer but severely inhibited binding to Cdc6. Conversely, deletion of N-terminal sequences within the conserved R3 domain blocked binding to the A-subunit but had no effect on interaction with Cdc6. The N-terminal region required for interaction with the A-subunit contains an FYF amino acid motif that plays a role in the interaction and is conserved between members of the PPP2R3 family. The A-subunit of PP2A is a HEAT repeat protein (36). The FYF motif in PR70 resembles the FG amino acid repeats (FXFG and GLFG) within the nucleoporin family of nuclear pore proteins. The nucleoporins interact with nuclear transport factors, including importin-β, which are also HEAT repeat proteins. The FG repeats of nucleoporins bind to shallow hydrophobic pockets in importin-β (37, 38). The A-subunit of PP2A contains exposed hydrophobic surfaces, predicted to play a role in interaction with the regulatory subunits (36), that are possible sites of interaction with the FYF motif of PR70.

The requirement for the N-terminal region of the R3 domain of PR70 for binding to the A-subunit is distinct from results observed with the PR72 protein. A fragment of PR72 consisting of amino acids 219-473 (corresponding to residues 257-509 of PR70) interacts with the A-subunit in the yeast two-hybrid assay (27). This fragment of PR72 is missing the N-terminal region of the R3 domain. Two fragments of PR72 containing putative A-subunit binding domains prepared by in vitro translation (corresponding to residues 234-339 and 378-436 of PR70) interacted with the A-subunit in vitro using GST pulldown assays (39). These PR72 fragments also do not contain the conserved N-terminal region of the R3 domain that was necessary for interaction of PR70 with the AC core dimer in our assays. These observations indicate that additional regions of PR70, beyond those required in PR72, are required for binding to the A-subunit, or that the differences observed are due to different assays employed.

In summary, the present study shows that the PR70 regulatory subunit targets PP2A to Cdc6 and that PP2A is likely to be a physiological Cdc6 phosphatase. The targeting of PP2A to Cdc6 is enhanced by binding of calcium to PR70 raising the possibility that changes in intracellular calcium can influence formation of pre-replicative complexes through regulation of Cdc6 dephosphorylation.


   FOOTNOTES
 
* This work was supported, in whole or in part, by National Institutes of Health Grant GM49505. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2. Back

1 Supported by National Institutes of Health Pharmacological Sciences Training Grant T32 GM07062. Back

2 To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-645-6152; Fax: 214-645-6151; E-mail: marc.mumby{at}utsouthwestern.edu.

3 The abbreviations used are: pre-RC, pre-replicative complex; PP2A, protein phosphatases 2A; APC/C, anaphase promoting complex/cyclosome; siRNA, small interfering RNA; Cdk, cyclin-dependent kinase; E3, ubiquitin-protein isopeptide ligase; PBS, phosphate-buffered saline; IP, immunoprecipitation; GST, glutathione S-transferase; aa, amino acid(s); HA, hemagglutinin; CMV, cytomegalovirus; Cdc6, cell division control protein 6. Back



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 ABSTRACT
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 DISCUSSION
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