Supplemental Material can be found at:
http://www.jbc.org/cgi/content/full/M710313200/DC1
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 23, pp. 16104 –16114, June 6, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Protein Phosphatase 2A Is Targeted to Cell Division Control
Protein 6 by a Calcium-binding Regulatory Subunit*□
S
Received for publication, December 19, 2007, and in revised form, February 29, 2008 Published, JBC Papers in Press, April 8, 2008, DOI 10.1074/jbc.M710313200
Anthony J. Davis‡1, Zhen Yan§, Bobbie Martinez‡, and Marc C. Mumby‡2
From the ‡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
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
* 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.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Figs. S1 and S2.
1
Supported by National Institutes of Health Pharmacological Sciences Training Grant T32 GM07062.
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@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, ubiquitinprotein 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.
16104 JOURNAL OF BIOLOGICAL CHEMISTRY
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 CDKmediated 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,
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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.
PR70 Targets PP2A to Cdc6
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
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JOURNAL OF BIOLOGICAL CHEMISTRY
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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), 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 ␮l 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 ⫻ g for 10
min, and protein complexes were immunoprecipitated from
the supernatant. Endogenous PR70 and Cdc6 were immunoprecipitated from 1.2 ⫻ 106 HeLa cells lysed in 300 ␮l 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 ␮l of antiserum and 40 ␮l
of protein A-Sepharose (Sigma-Aldrich) were added to 300 ␮l
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 ␮l of 2⫻ 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 ⫻ 106 cells using 7 ␮g of anti-FLAG polyclonal antibody (Sigma-Aldrich) or 7 ␮g of non-immune rabbit
IgG (Sigma-Aldrich) and 40 ␮l of protein A-Sepharose (SigmaAldrich) for 2 h at 4 °C. The immunoprecipitates were washed
three times with lysis buffer and solubilized in 60 ␮l of 2⫻
SDS-PAGE loading buffer. 30 ␮l 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
PR70 Targets PP2A to Cdc6
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Cloning of GST-tagged PR70 and EF-hand Mutants—PR70
and PR70 EF-hand mutant cDNA were cloned into the pGEX4T-1 vector (Amersham Biosciences). The cDNAs were amplified by PCR using the pCMV-Tag2B vector containing the fulllength 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 GSTPR70 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 ⫻ 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 ⫻ 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 ␮l of IP lysis buffer or in 300 ␮l of IP
lysis buffer containing 10 mM EDTA or 10 mM CaCl2 for 20 min.
GST pulldown assays (26) were conducted by incubating 300 ␮l
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 ␮M 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 ␮l of 2⫻ SDS-PAGE
loading buffer. 30 ␮l 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
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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
␮Ci/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 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 EFhands 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 ⌬N1 (aa 125–
575) corresponds to the PR48 protein described previously
(17). ⌬N2, ⌬N3, and ⌬C were generated using the following
primers: ⌬N2 (aa 136 –575) 5⬘-CGGGATCCGCCACCATGGATGACATG-3⬘, ⌬N3 (aa 162–575) 5⬘-CGGGATCCAGGACTCCGTCAACGTG-3⬘, and ⌬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.
PR70 Targets PP2A to Cdc6
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 SDSPAGE 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⌬N mutant (⌬N1), full-length FLAG-PR70, or a
JUNE 6, 2008 • VOLUME 283 • NUMBER 23
RESULTS
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
combination of plasmids expressing FLAG-PR70 and HA-Cdc6 (Cdc6). Cells
were harvested after 24 h, and lysates were immunoprecipitated with antiFLAG 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.
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(M2, Stratagene), anti-PP2A C-subunit 1F6, and anti-PP2A
A-subunit (C-20, Santa Cruz Biotechnology) antibodies and
developed as described above.
Flow Cytometry—U2OS cells (4 ⫻ 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
␮g/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.
PR70 Targets PP2A to Cdc6
16108 JOURNAL OF BIOLOGICAL CHEMISTRY
A
EF1
EF2
PR70
45
EF1/EF2(-z)
EF1/EF2(x,y)
EF2(-z)
EF2(x,y)
EF1(-z)
PR70
B
EF1(x,y)
X Y Z-X-Y -Z
X Y Z-X-Y -Z
WT
166-DTDHDLLIDADD-177 240-DLDGDGALSMFE-251
ATAHDLLIDADD
EF1(x,y)
DLDGDGALSMFE
EF1(-z)
DTDHDLLIDADN
DLDGDGALSMFE
EF2(x,y)
DTDHDLLIDADD
ALAGDGALSMFE
EF2(-z)
DTDHDLLIDADD
DLDGDGALSMFQ
EF1/EF2(x,y) ATAHDLLIDADD
ALAGDGALSMFE
EF1/EF2(-z) DTDHDLLIDADN
DLDGDGALSMFQ
Ca2+
CBB
C
GST-A
GST-Cdc6
N
E
Ca
Ca+CP
N
N
E
Ca
Ca+CP
GST
N
GST
1
2
3
4
5
6
7
8
9
10
FLAG-PR70
A
C
GST
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 EFhand 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 ␮M) 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-Asubunit (A), anti-C-subunit (C), and anti-GST (GST) antibodies.
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 GSTCdc6 (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
VOLUME 283 • NUMBER 23 • JUNE 6, 2008
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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 antiC-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.
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 EFhand (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 ␮M
(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
PR70 Targets PP2A to Cdc6
Ca
E
A
Ca
GST-A
pulldown
C
B
FL-PR70
GST-Cdc6
pulldown
A
7
8
9
EF1/EF2 (x,y)
EF1/EF2(-z)
5
6
EF2(x,y)
4
EF1(-z)
3
EF1(x,y)
Emp Vec
2
PR70
1
EF2(-z)
C
PR70
Conserved R3 domain unique
FYF motif
EF1 EF2
PR70
∆N1
∆N2
∆N3
∆C
B
Emp Vec
FL-PR70
C
PR70
unique
1-575
125-575
136-575
162-575
1-441
∆C
E
∆N3
E Ca
∆N2
EF2(-z) EF1/2(-z)
EF1(-z)
E Ca
∆N1
N
PR70
GST PR70
A
FLAG
FL-PR70
A
A OE
C OE
1
2
3
4
5
6
7
8
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 EFhand 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 antiA-subunit (A OE) and anti-C-subunit (C OE) immunoblots are also shown.
loss of interaction with PP2A (Fig. 3C, lanes 5– 8). A longer
exposure of the blot showed that a weak interaction of the Aand 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.
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 ⌬N1
mutant contains a deletion of the entire N-terminal PR70unique region and interacted with endogenous PP2A subunits
to the same extent as full-length PR70 (Fig. 4B). Deletion of a
JUNE 6, 2008 • VOLUME 283 • NUMBER 23
A
C
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-Csubunit (C) antibodies. A control immunoprecipitate using lysate from cells
transfected with the empty expression vector (Emp Vec) is shown in the first
lane.
C-terminal segment that included the PR70-unique region
(⌬C) had little, if any, effect on the interaction with PP2A. However, deletions of N-terminal regions of the conserved R3
domain, ⌬N2 and ⌬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 ⌬N3, and the ⌬C mutants in the presence and absence of
calcium. As expected, full-length PR70 and the ⌬C mutant
interacted with the A-subunit of PP2A, whereas the ⌬N3
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Anti-FLAG
IP
C
Anti-FLAG
IP
PR70 Targets PP2A to Cdc6
A
Human PR70
Human PR72
Mouse PR59
Xenopus PR70
Drosophila PR72
IPTFYFPRGRP
IPRFYFGEGLP
VPAFYFPCGRP
IPKFYFPKGCP
IPRFYFPHGKP
FYF motif
EF1 EF2
B
PR70
AAA
AYA
FYA
FAF
AYF
DN3
FLAG
Anti-FLAG
IP
A
C
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 ⌬N3 mutant (⌬N3), and
the indicated FYF mutants were transiently expressed in COS-7 cells. The
cells were lysed, and tagged proteins were immunoprecipitated with antiFLAG 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.
A
∆N3
PR70
GST E
Ca GST E
∆C
Ca GST E
Ca
FLAG
GST-A
pulldown
C
B
FLAG
GST-Cdc6
pulldown
AC
1
2
3
4
5
6
7
8
9
FIGURE 6. The C-terminal region of PR70 mediates interaction with
Cdc6. FLAG-PR70 (lanes 1–3), the ⌬N3 mutant (lanes 4 – 6), and the ⌬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).
16110 JOURNAL OF BIOLOGICAL CHEMISTRY
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Emp Vec
C
PR70
WT 125-IPTFYFPRGRP-135
IPTAYFPRGRP
AYF
IPTFAFPRGRP
FAF
IPTFYAPRGRP
FYA
IPTAYAPRGRP
AYA
IPTAAAPRGRP
AAA
mutant did not (Fig. 6A). Furthermore, there was an enhanced
interaction of PR70 and the ⌬C mutant in the presence of calcium. As shown previously with full-length PR70 (Fig. 2C), the
enhanced binding of PR70 and the ⌬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 ⌬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.
Both full-length PR70 and the ⌬N3 mutant bound to GSTCdc6 (Fig. 6B). However, only full-length PR70 was able to
recruit additional A- and C-subunits in the presence of calcium.
In contrast, the ⌬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 ⌬N3 or ⌬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 ⌬N3 or ⌬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
PP2A-C
Em
Cdc6
pt
y
V
C
dc ec
to
6
r
C
dc
6
C +C
dc
D
K2
6
+
C
dc PR
6
70
C +P
dc
R
70
6
+
∆N
C
P
dc
3
R
70
6
+
∆
PR C
70
EF
1/
2
A
PP5
PP2A-C
siRNA
Luc
A
Mock
PR70 Targets PP2A to Cdc6
GFP-Cdc6
PR70
Endo-Cdc6
FLAG-PR70
PR70-2
PR70-1
siRNA
Luc
B
Mock
PP5
Myc-CDK2
D
plasmid
EV
PR70
Luc
PR70
Luc
PR70
Luc
+
PR
6
D
D
D
-C
dc
6
dc
-C
D
D
D
A-
C
dc
6
6
+
PR
70
A-
C
dc
+
6
dc
AA
AA
r
6
tC
dc
tC
GFP-Cdc6
GFP-Cdc6
Endo-Cdc6
GPDH
FLAG-PR70
Actin
wtCdc6 DDD-Cdc6 AAA-Cdc6
Luc siRNA
PR70 siRNA
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 antiCdc6, 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.
mutated to non-phosphorylatable alanine residues (AAACdc6). 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.
JUNE 6, 2008 • VOLUME 283 • NUMBER 23
w
wtCdc6 DDD-Cdc6 AAA-Cdc6
PR70
Luc
siRNA
EV
Em
C
plasmid
w
pt
y
Ve
Actin
PR
70
B
ct
o
PR70
70
Actin
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 wildtype 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 ⌬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 ⌬C
mutant, which binds to the AC core dimer but not to Cdc6, had
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Cdc6
PR70 Targets PP2A to Cdc6
A
500
Non-transfected
G0/G1 52.1
400
S 22.2
300
G2/M 24.4
200
100
0
B
Luc control
G0/G1 55.3
400
S 22.2
300
No. of Cells
100
0
C
PR70 siRNA-2
G0/G1 65
600
S 16.3
400
G2/M 17.9
200
0
800
D
PR70 siRNA-1
G0/G1 76.5
600
S 12.1
400
G2/M 10.7
200
200
400
600
FL3 Area
800
1
A-
si
PR
70
si
nt
R
ro
R
N
l
ec
co
c
an
sf
Lu
-tr
N
on
N
A-
2
te
d
E
70
0
PR
0
PR70
DISCUSSION
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
GPDH
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
16112 JOURNAL OF BIOLOGICAL CHEMISTRY
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.
VOLUME 283 • NUMBER 23 • JUNE 6, 2008
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G2/M 20.9
200
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”).
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.
PR70 Targets PP2A to Cdc6
JUNE 6, 2008 • VOLUME 283 • NUMBER 23
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 prereplicative 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 famJOURNAL OF BIOLOGICAL CHEMISTRY
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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␥, 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 (⌬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 ⌬N3 and EF1/2 mutants) but are
dependent on interaction with Cdc6 (e.g. the ⌬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 ⌬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
PR70 Targets PP2A to Cdc6
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ily. 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-␤, which are also HEAT repeat proteins.
The FG repeats of nucleoporins bind to shallow hydrophobic
pockets in importin-␤ (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.