Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html
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
The cell division control protein 6 (Cdc6) is essential for for-
cyclin-dependent kinases (CDKs) and the E3 ubiquitin ligase,
mation of pre-replication complexes at origins of DNA replica-
anaphase promoting complex/cyclosome (2). The mammalian
tion. Phosphorylation of Cdc6 by cyclin-dependent kinases
Cdc6 protein is required for DNA replication and acts in con-
Downloaded from
inhibits ubiquitination of Cdc6 by APC/Ccdh1 and degradation
junction with the Cdt1 protein to recruit the mini-chromosome
by the proteasome. Experiments described here show that the
maintenance complex into pre-RCs (1, 3, 4). Mammalian cells
PR70 member of the PPP2R3 family of regulatory subunits tar-
have multiple mechanisms to ensure that pre-RCs only assem-
gets protein phosphatase 2A (PP2A) to Cdc6. Interaction with
ble during late M and G , including regulation of the levels and
1
Cdc6 is mediated by residues within the C terminus of PR70,
function of Cdc6 (2).
www.jbc.org
whereas interaction with PP2A requires N-terminal sequences
Mammalian Cdc6 is regulated by phosphorylation of multi-
conserved within the PPP2R3 family. Two functional EF-hand
ple sites within its N-terminal domain by cyclin-dependent
calcium-binding motifs mediate a calcium-enhanced interac-
protein kinases. Cdc6 is phosphorylated at canonical CDK sites,
tion of PR70 with PP2A. Calcium has no effect on the interac-
including serines 54, 74, and 106 of human Cdc6 (5, 6). Exper-
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
tion of PR70 with Cdc6 but enhances the association of PP2A
iments with exogenously expressed protein have shown that
with Cdc6 through its effects on PR70. Knockdown of PR70 by
phosphorylation can regulate the nuclear localization of Cdc6
RNA interference results in an accumulation of endogenous and
(5, 7–9). However, other studies have shown that a subpopula-
expressed Cdc6 protein that is dependent on the cyclin-depend-
tion of endogenous Cdc6 remains in the nucleus, bound to
ent protein kinase phosphorylation sites on Cdc6. Knockdown
chromatin, throughout the cell cycle (10 –12). Phosphorylation
of PR70 also causes G1 arrest, suggesting that PR70 function is
of Cdc6 also plays an important role in regulating the stability of
critical for progression into S phase. These observations indi-
Cdc6. The N-terminal domain of Cdc6 contains RXXL (D box)
cate that PP2A can be targeted in a calcium-regulated manner to
and KEN (KEN box) destruction motifs, which are binding sites
Cdc6 via the PR70 subunit, where it plays a role in regulating
for the form of the APC/C containing the cdh1-targeting sub-
protein phosphorylation and stability.
unit (13). Cdc6 is polyubiquitinated and targeted for degrada-
tion by APC/Ccdh1, which prevents formation of pre-RCs in
quiescent cells and during early G by maintaining low levels of
1
Precise regulation of DNA replication is necessary to ensure
Cdc6 (14). Phosphorylation of Cdc6 by CDKs protects the pro-
that daughter cells receive a complete and intact genome dur-
tein from degradation by blocking recognition by cdh1 result-
ing mitosis. A crucial step in regulating DNA replication is the
ing in stabilization of Cdc6 during a window of time that allows
assembly of pre-replicative complexes at origins of replication
formation of pre-RCs during G (15). The importance of CDK-
1
(1). Coordination of DNA replication with the cell cycle is
mediated stabilization of Cdc6 is also supported by evidence
achieved through a periodic accumulation and destruction of
showing that the cell cycle arrest caused by DNA damage is due
proteins involved in formation of pre-RCs3 is mediated by
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
* 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
phosphatases, dephosphorylation of Cdc6 can also control for-
part by the payment of page charges. This article must therefore be hereby
mation of pre-RCs. Much less is known about mechanisms that
marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to
regulate Cdc6 dephosphorylation. A previous study identified a
indicate this fact.
□
S The on-line version of this article (available at http://www.jbc.org) contains
fragment of PR70 as a member of the PPP2R3 family of PP2A
supplemental Table S1 and Figs. S1 and S2.
regulatory subunits that interacted with Cdc6 and implicated
1 Supported by National Institutes of Health Pharmacological Sciences Train-
PP2A in regulating Cdc6 phosphorylation (17). The major
ing Grant T32 GM07062.
2 To whom correspondence should be addressed: Dept. of Pharmacology,
forms of PP2A contain a dimeric core complex composed of a
University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd.,
scaffold (A) and a catalytic subunit (C). The AC core dimer
Dallas, TX 75390-9041. Tel.: 214-645-6152; Fax: 214-645-6151; E-mail:
associates with regulatory subunits that form heterotrimeric
marc.mumby@utsouthwestern.edu.
3 The abbreviations used are: pre-RC, pre-replicative complex; PP2A, protein
holoenzymes and target the catalytic subunit to specific phos-
phosphatases 2A; APC/C, anaphase promoting complex/cyclosome;
phoprotein substrates (18 –20). In this study, the mechanism
siRNA, small interfering RNA; Cdk, cyclin-dependent kinase; E3, ubiquitin-
and functional consequences of targeting of PP2A to Cdc6 by
protein isopeptide ligase; PBS, phosphate-buffered saline; IP, immunopre-
PR70 were investigated. The results show that PR70 interacts
cipitation; GST, glutathione S-transferase; aa, amino acid(s); HA, hemagglu-
tinin; CMV, cytomegalovirus; Cdc6, cell division control protein 6.
with PP2A and Cdc6 through distinct regions of the protein,
16104 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 283 • NUMBER 23 • JUNE 6, 2008
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html
PR70 Targets PP2A to Cdc6
that the association of PP2A to Cdc6 is enhanced by calcium
Immunoprecipitation and Immunoblotting—Rabbit antisera
binding to PR70, and that loss of PR70 causes increased levels of
were raised against a synthetic peptide corresponding to the C
Cdc6 and G arrest.
terminus of human PR70 (CDLYEYACGDEDLEPL) conju-
1
gated to keyhole limpet hemocyanin. Anti-PR70 antibodies
EXPERIMENTAL PROCEDURES
were affinity purified on a peptide column made with the same
Cloning of Full-length PR70—A human expressed sequence
peptide using the MicroLink Peptide Coupling Kit (Pierce) fol-
tag encoding the PR70 start codon was identified in the human
lowing the manufacturer’s protocol. Rabbit antiserum against
expressed sequence tag data base using the MegaBLAST tool
human Cdc6 was generated against a full-length Cdc6 fusion
protein as described previously (4).
(www.ncbi.nlm.nih.gov/BLAST/) with the assembled PR70
Proteins were immunoprecipitated following the protocol
sequence (21). A PR70 cDNA was constructed using the PR48
described previously (17). Briefly, the media was aspirated and
cDNA and the IMAGE Human Clone ID 5728169 (GenBankTM
the cells were washed with cold PBS. The cells were incubated
accession number BM544432), purchased from Invitrogen,
on ice for 20 min in 300 l of IP lysis buffer containing 20 mM
using an internal NcoI restriction site present in the common
Tris-HCl (pH 7.5), 0.2% Nonidet P-40, 20% glycerol, 200 m
Downloaded from
M
region of BM54432 and PR48. A PCR fragment containing the
NaCl, 1 mM EDTA, and protease inhibitor mixture (Roche
translational start codon, the 5Ј-end, and the 3Ј-NcoI site of
Applied Science). Lysates were centrifuged at 14,000 ϫ g for 10
BM54432 was generated using the BM54432 cDNA as template
min, and protein complexes were immunoprecipitated from
with the PCR primers: 5Ј-CGGGATCCATGCCGCCCGGCA-
the supernatant. Endogenous PR70 and Cdc6 were immuno-
AAGT-3Ј (sense strand) and 5Ј-GCGCCTTGATCCGGC-3Ј
precipitated from 1.2 ϫ 106 HeLa cells lysed in 300 l of IP lysis
(antisense strand). The PCR product was digested with the
www.jbc.org
buffer as described above. PR70 was immunoprecipitated using
restriction enzymes BamHI and NcoI. The 3Ј portion of the
a rabbit antiserum generated against the peptide CDLYEY-
PR48 cDNA was excised from the PR48 cDNA (17) using NcoI
ACGDEDLEPL conjugated to hemocyanin. Cdc6 was immuno-
and HindIII, and the fragments were ligated and subcloned into
precipitated using a rabbit polyclonal antibody generated
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
the pCMV-Tag2B vector (Stratagene) digested with BamHI
against a full-length Cdc6-GST fusion protein described previ-
and HindIII. The resulting construct encoded a full-length
ously. As a negative control, immunoprecipitations were per-
PR70 cDNA fused to an N-terminal FLAG epitope tag. The
formed using pre-immune serum collected from the rabbits
sequence was verified by automated sequencing.
immunized against PR70 or Cdc6. 10 l of antiserum and 40 l
Cell Culture, Transfection, and RNA Interference—COS-7,
of protein A-Sepharose (Sigma-Aldrich) were added to 300 l
HeLa, and U2OS cells were maintained at 37 °C in Dulbecco’s
of lysate, and the mixture was incubated for 2 h at 4 °C. The
modified Eagle’s medium containing 10% fetal bovine serum in
protein-A beads were washed three times with IP lysis buffer,
an atmosphere of 5% CO . U2OS, obtained from the ATCC, is a
2
and protein was solubilized in 60 l of 2ϫ SDS-PAGE loading
human osteosarcoma cell line that expresses wild-type p53. For
buffer. Thirty microliters of solubilized material was resolved
transient expression of proteins, cells were transfected with
on a 10% SDS-PAGE gel and transferred to a nitrocellulose
expression plasmids using Lipofectamine 2000 (Invitrogen)
membrane. The membrane was cut into pieces, which were
according to the manufacturer’s protocol. Cells were harvested
probed with anti-PP2A C-subunit monoclonal antibody 1F6
either 24 or 48 h after transfection. Transfection with small
(24), anti-PP2A A-subunit antiserum (C-20, Santa Cruz Bio-
interfering RNA to knock down PR70 was carried out using
technology), anti-Cdc6 monoclonal antibody (clone DCS-180,
Oligofectamine (Invitrogen) following the manufacturer’s pro-
Upstate), or anti-PR70 antiserum. Following incubation with
tocol. Annealed duplex siRNAs were purchased from Dharma-
horseradish peroxidase-conjugated secondary antibodies, the
con and had the following sequences: PR70-1, 5Ј-AGCCGG-
blots were developed using the enhanced chemiluminescence
UCCUGAAGAUGAAdTdT-3Ј (sense strand) and PR70-2,
detection system (Amersham Biosciences).
5Ј-AAAGCAUUCCGACCUUCUAdTdT-3Ј (sense strand).
Transiently expressed FLAG-tagged proteins were immuno-
Controls included an siRNA that knocks down the MEKK2
precipitated from 1.5 ϫ 106 cells using 7 g of anti-FLAG poly-
protein kinase (22), an siRNA that knocks down protein phos-
clonal antibody (Sigma-Aldrich) or 7 g of non-immune rabbit
phatase 5 (23), and an siRNA corresponding to the sequence of
IgG (Sigma-Aldrich) and 40 l of protein A-Sepharose (Sigma-
firefly luciferase (5Ј-TCGAAGTATTCCGCGTACGdTdT-3Ј).
Aldrich) for 2 h at 4 °C. The immunoprecipitates were washed
Cells were lysed and analyzed by immunoblotting 48 h after
three times with lysis buffer and solubilized in 60 l of 2ϫ
transfection. In some experiments, cells were co-transfected
SDS-PAGE loading buffer. 30 l of solubilized protein was
with PR70 siRNA and expression plasmids encoding wild-type
resolved on a 10% SDS-PAGE gel and transferred to a nitrocel-
Cdc6 or Cdc6 mutants in which all three N-terminal phospho-
lulose membrane. The membrane was probed with anti-FLAG
rylation sites were mutated to alanine (AAA-Cdc6) or aspartic
M2 monoclonal (Stratagene), anti-PP2A C-subunit 1F6, and
acid (DDD-Cdc6) using Lipofectamine 2000 and harvested 48 h
anti-PP2A A-subunit (C-20, Santa Cruz Biotechnology) anti-
later. The cDNAs encoding phosphorylation site mutants of
bodies and developed as described above.
Cdc6 were prepared using a PCR-based site-directed mutagen-
Calcium Overlay Assay—In vitro 45Ca2ϩ overlay assays were
esis kit (Invitrogen) according to the manufacturer’s protocol.
carried out using a protocol described previously (25). Purified
Wild-type and mutant Cdc6 were expressed as a fusion proteins
GST fusion proteins were resolved by SDS-PAGE and trans-
fused to the N terminus of enhanced green fluorescent protein
ferred to a nitrocellulose membrane. The membrane was
using the pEGFP-N expression vector (Clontech).
washed three times in IMK buffer (10 mM imidazole-HCl, pH
JUNE 6, 2008 • VOLUME 283 • NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 16105
Supplemental Material can be found at:
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PR70 Targets PP2A to Cdc6
6.8, 5 mM MgCl , and 60 m
Cloning of GST-tagged PR70 and EF-hand Mutants—PR70
2
M KCl) for 1 h at room temperature.
The membrane was then incubated in IMK buffer containing 5
and PR70 EF-hand mutant cDNA were cloned into the pGEX-
Ci/ml 45Ca2ϩ for 10 min. The membrane was washed three 4T-1 vector (Amersham Biosciences). The cDNAs were ampli-
times in 30% ethanol for 5 min, dried, and exposed to x-ray film
fied by PCR using the pCMV-Tag2B vector containing the full-
for 12 h.
length PR70 or EF-hand mutant cDNA as template with the
Generation of PR70 Mutants—Point mutations were intro-
following primers: 5Ј-CGGGATCCATGCCGCCCGGCAA-
duced into the EF-hands of PR70 as described in the manual for
AGT-3 (sense strand) and 5Ј-ATTTGCGGCCGCTCACAG-
the QuikChange Multi Site-directed mutagenesis kit (Strat-
CGGCTCCAGGTC-3Ј (antisense strand). The products were
agene) using the following primers (mutated residues underlined).
digested with BamHI and NotI and ligated into pGEX 4T-1,
PCR was performed with pCMV-Tag2B containing the full-length
which had been cut with the same restriction enzymes. The
PR70 cDNA as template with the following primers: EF1(x,y) 5Ј-
resulting constructs encode the GST protein fused to the N
CAAGTTCTGGGAGCTGGCCACGGCCCACGACCTGCTC-
terminus of full-length PR70 or EF-hand mutant proteins. The
ATCG-3Ј (sense strand) and 5Ј-CGATGAGCAGGTCGTGGG-
sequences were verified by automated sequencing.
Downloaded from
CCGTGGCCAGCTCCCAGAACTTG-3Ј (antisense strand),
Expression and Purification of GST-Cdc6, GST-A, and GST-
EF1(-z) 5Ј-TTGTGCCGCGCCAGGTTGTCCGCGTCGATGA-
PR70 Fusion Proteins—A GST-Cdc6 fusion protein was pre-
GCGCTCATCGACGCGGACAACCTGGCGCGGCACAA-3Ј
pared by a modification of a method previously described (6).
(sense strand) and 5Ј 3 3Ј (antisense strand), EF2(x,y) 5Ј-TGGT-
Briefly, 1 liter of Sf9 cells (2 ϫ 106 cells/ml) was infected with
TCCGCTGCATGGCCCTGGCCGGGGACGGCGCCCTG-3Ј
recombinant GST-Cdc6 baculovirus (a gift of Dr. Ellen Fan-
(sense strand) and 5Ј-CAGGGCGCCGTCCCCGGCCAGGGC-
ning, Vanderbilt University) at a Sf9 culture:baculovirus ratio of
www.jbc.org
CATGCAGCGGAACCA-3Ј (antisense strand), and EF2(-z), 5Ј-
1:20 (v/v) for 60 h. The cells were collected by centrifugation
GCGCCCTGTCCATGTTCCAGCTCGAGTACTTCTAC-3Ј
and washed once with PBS. Cells were lysed on ice in 40 ml of
(sense strand) and 5Ј-GTAGAAGTACTCGAGCTGGAACAT-
buffer A (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM KCl,
0.5 m
, 0.5% Nonidet P-40, 1 m
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
GGACAGGGCGC-3Ј (antisense strand). Mutations in both EF-
M MgCl2
M dithiothreitol, 10 mM
NaF, 1 m
hands were introduced using the EF1 mutant cDNAs as tem-
M EGTA, 2 mM EDTA, and a protease inhibitor tablet
(Roche Applied Science)) using a Dounce homogenizer. Lysates
plate for PCR with primers for introduction of EF2 point
were centrifuged at 30,000 ϫ g for 30 min at 4 °C to remove
mutants. All mutations were verified by automated sequencing.
cellular debris, and the lysate was mixed with 2 ml of glutathi-
PR70 truncation mutants were generated by PCR amplifi-
one-agarose (Sigma-Aldrich) for 2 h at 4 °C. The resin was
cation using the PR70 cDNA as template. The ⌬N1 (aa 125–
recovered by centrifugation and washed twice with PBS, once
575) corresponds to the PR48 protein described previously
with PBS containing 1.5 M NaCl, and once with PBS containing
(17). ⌬N2, ⌬N3, and ⌬C were generated using the following
1.5 mM NaCl and 0.1% (v/v) Nonidet P-40, and then re-equili-
primers: ⌬N2 (aa 136 –575) 5Ј-CGGGATCCGCCACCATG-
brated in PBS. The GST-Cdc6 fusion protein immobilized on
GATGACATG-3Ј, ⌬N3 (aa 162–575) 5Ј-CGGGATCCAG-
glutathione agarose beads was resuspended in buffer B (20 mM
GACTCCGTCAACGTG-3Ј, and ⌬C (aa 1– 441) 5Ј-CCCA-
HEPES, pH 7.6, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA,
AGCTTCATCTGGCAGAGGCAGTC-3Ј.
and 50% glycerol) and stored at Ϫ80 °C.
Point mutations were introduced into the FYF motif (aa
GST-A fusion protein, GST-PR70, and GST-EF-hand
128 –130) of PR70 using the full-length PR70 cDNA as template
mutants were expressed in bacteria and prepared as previously
with the following mutagenic primers (mutated residues under-
described (26). The GST fusion proteins immobilized on gluta-
lined): AYF, 5Ј-GCCAAAGCATTCCGACCGCCTACTTCCC-
thione agarose beads were resuspended in buffer B and stored
CAGAGGACG-3Ј (sense strand) and 5Ј-CGTCCTCTGGGGA-
at Ϫ80 °C until use.
AGTAGGCGGTCGGAATGCTTTGGC-3Ј (antisense strand),
GST Pulldown Assays—GST, GST-A, and GST-Cdc6 immo-
FAF, 5Ј-CCAAAGCATTCCGACCTTCGCCTTCCCCAGAG-
bilized on glutathione-agarose beads were used to assess the
GACGCC-3Ј (sense strand) and 5Ј-GGCGTCCTCTGGGGAA-
binding of PR70. Wild-type or mutant FLAG-PR70 was
GGCGAAGGTCGGAATGCTTTGG-3Ј (antisense strand),
expressed by transient transfection of COS-7 cells. The cells
FYA, 5Ј-GCATTCCGACCTTCTACGCCCCCAGAGGACGC-
were lysed on ice in 300 l of IP lysis buffer or in 300 l of IP
CCGC-3Ј (sense strand) and 5Ј-GCTTTCGTCCTCTGGGGGC-
lysis buffer containing 10 mM EDTA or 10 mM CaCl for 20 min.
2
GTAGAAGGTCGGAATGC-3Ј (antisense strand). To make the
GST pulldown assays (26) were conducted by incubating 300 l
AYAP mutant, the AYFP cDNA was used as a template, and
of lysate with either GST, GST-A, or GST-Cdc6. The samples
PCR mutagenesis was done with the following primers: AYA,
were incubated for 1 h at room temperature with agitation. The
5Ј-CATTCCGACCGCCTACGCCCCCAGAGGACGCCCG-3Ј
calpain inhibitor calpeptin (Calbiochem) was added at a con-
(sense strand) and 5Ј-CGGGCGTCCTCTGGGGGCGTAGGC-
centration of 50 M in some experiments. Following incuba-
GGTCGGAATG-3Ј (antisense strand). To make the AAAP
tion, the sample was washed three times with IP lysis buffer
mutant, the AYAP cDNA was used as a template, and PCR
supplemented with EGTA, CaCl , or CaCl and calpeptin, and
2
2
mutagenesis was done with the following primers AAA, 5Ј-GCA-
the beads were collected by centrifugation. After washing, the
TTCCGACCGCCGCCGCCCCCAGAGGACG-3Ј
(sense
bound proteins were solubilized in 60 l of 2ϫ SDS-PAGE
strand) and 5Ј-CGTCCTCTGGGGGCGGCGGCGGTCGG-
loading buffer. 30 l of solubilized protein was resolved on a
AATGC-3Ј (antisense strand). All mutations were verified
10% SDS-PAGE gel and transferred to a nitrocellulose mem-
by automated sequencing.
brane. The membrane was probed with anti-FLAG monoclonal
16106 JOURNAL OF BIOLOGICAL CHEMISTRY
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Supplemental Material can be found at:
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PR70 Targets PP2A to Cdc6
(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 ϫ 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
Downloaded from
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 fig-
www.jbc.org
ures are from individual experiments that were representative
of common results obtained in at least three independent
experiments.
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
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 termi-
nus of PR70 and affinity purified on a peptide column. The
purified antibodies recognized a protein band of M ϭ 70,000 in
r
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
FIGURE 1. PR70 interacts with Cdc6 and PP2A. A, Cdc6 was immunoprecipi-
lysates were immunoprecipitated with PR70 and Cdc6 antibod-
tated from exponentially growing HeLa cells using a polyclonal antiserum
ies. Immunoprecipitation of Cdc6 co-precipitated a diffuse
specific for Cdc6 (Cdc6) or pre-immune serum (Pre). The immunoprecipitates
protein band that migrated at the position of PR70 that was not
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-
combination of plasmids expressing FLAG-PR70 and HA-Cdc6 (Cdc6). Cells
PAGE and analyzed by immunoblotting with anti-PR70 (PR70), anti-A-subunit
were harvested after 24 h, and lysates were immunoprecipitated with anti-
(A), and anti-C-subunit (C) antibodies as indicated on the left. C, HeLa cells
FLAG antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE
were transiently transfected with empty expression vector (Emp Vec), plas-
and immunoblotted with anti-FLAG (FLAG-PR70), anti-Cdc6, anti-A-subunit,
mids expressing the FLAG-PR70⌬N mutant (⌬N1), full-length FLAG-PR70, or a
and anti-C-subunit antibodies as indicated on the left.
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PR70 Targets PP2A to Cdc6
present in immunoprecipitates obtained with the preimmune
serum (Fig. 1A). The anti-Cdc6 serum also co-precipitated the
A
EF1
EF2
A- and C-subunits of PP2A. Although the anti-PR70 antibodies
PR70
co-precipitated the A- and C-subunits of PP2A (Fig. 1B), a com-
plex between PR70 and Cdc6 could not be detected. The inabil-
X Y Z-X-Y -Z
X Y Z-X-Y -Z
ity to detect Cdc6 may be due to steric hindrance by the anti-
WT
166-DTDHDLLIDADD-177 240-DLDGDGALSMFE-251
EF1(x,y)
ATAHDLLIDADD
DLDGDGALSMFE
C-terminal antibody, because the C terminus of PR70 is
EF1(-z)
DTDHDLLIDADN
DLDGDGALSMFE
required for interaction with Cdc6 (see below). The association
EF2(x,y)
DTDHDLLIDADD
ALAGDGALSMFE
EF2(-z)
DTDHDLLIDADD
DLDGDGALSMFQ
of PR70 with PP2A and Cdc6 was also tested in co-immunopre-
EF1/EF2(x,y) ATAHDLLIDADD
ALAGDGALSMFE
cipitation experiments using exogenously expressed pro-
EF1/EF2(-z)
DTDHDLLIDADN
DLDGDGALSMFQ
teins. FLAG-tagged PR70 was expressed in HeLa cells and
immunoprecipitated with anti-FLAG antibodies. Analysis of
B
the immunoprecipitates by immunoblotting showed that the
x,y)
endogenous A- and C-subunits of PP2A and HA-tagged
Downloaded from
PR70
EF1(
EF1(-z)
EF2(x,y)
EF2(-z)
EF1/EF2(x,y)
EF1/EF2(-z)
Cdc6 co-precipitated with FLAG-PR70 (Fig. 1C). These
45Ca2+
results indicate that PR70 can interact with both the PP2A
core dimer and Cdc6 in intact cells.
CBB
Calcium Enhances the Interaction of PR70 with the AC Core
GST
GST-A
GST
GST-Cdc6
C
Dimer and Recruits PP2A to Cdc6—Analysis of the amino acid
www.jbc.org
sequence of PR70 identified two EF-hand calcium binding
motifs that are conserved within the PPP2R3 family (supple-
N
N
E
Ca
Ca+CP
N
N
E
Ca
Ca+CP
mental Fig. S1). The roles of these motifs were tested in a gel
FLAG-PR70
overlay assay with wild-type PR70 and PR70 containing inacti-
A
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
vating mutations of the EF-hand motifs. Point mutants were
C
constructed that had substitutions of amino acids involved in
GST
calcium binding (28), including alanine substitutions at both
1
2
3
4
5
6
7
8
9
10
the x and y coordinates and a conservative change at the -z
FIGURE 2. Interaction of PR70 with PP2A is enhanced by calcium-binding.
coordinate (Fig. 2A). Wild-type PR70 bound calcium in the in
A, the figure shows a diagram of the location and sequences of wild-type
vitro 45Ca2ϩ overlay assay (Fig. 2B). Mutation of the first EF-
PR70 (WT) and PR70 mutants containing substitutions of calcium-binding
hand (EF1) resulted in reduced binding of calcium compared
residues within the EF-hand motifs (EF mutants). The canonical EF-hand resi-
dues involved in coordination of calcium are indicated by the letters x, y, z, -x,
with wild-type PR70. Mutation of the second EF-hand (EF2)
-y, and -z using standard nomenclature (28). The x, y, and -z residues that were
severely reduced calcium binding, whereas the double muta-
mutated are shown in bold type. B, GST fusions of wild-type PR70 (PR70) and
tion of EF1 and EF2 nearly abolished the ability of PR70 to bind
the EF-hand mutant were analyzed for calcium binding by 45Ca2ϩ overlay
assay. The amounts of GST-PR70 in each lane were determined by staining the
calcium.
gel with Coomassie Brilliant Blue (CBB). C, calcium enhances binding of PR70
Calcium binding causes a conformational change in the PR72
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
member of the PPP2R3 family that is associated with enhanced
lysed in standard buffer (N) or lysis buffer containing EGTA (E) or CaCl (Ca).
2
interaction with the A-subunit of PP2A (27). Therefore, the
Calpeptin (50 M) was included in some experiments (CaϩCP). The lysates
effects of calcium on the interaction of PR70 with PP2A and
were incubated with GST alone (GST), GST-A, or GST-Cdc6, and bound pro-
teins were detected by immunoblotting with anti-FLAG (FLAG-PR70), anti-A-
Cdc6 were determined. FLAG-tagged PR70 was expressed in
subunit (A), anti-C-subunit (C), and anti-GST (GST) antibodies.
COS-7 cells, which were lysed in buffer containing EGTA or
calcium. The lysates were incubated with either GST-A or
addition of calcium resulted in enhanced binding of wild-type
GST-Cdc6 and bound proteins detected by immunoblotting.
PR70 and the EF1 mutant to GST-A, but not to GST-Cdc6 (Fig.
FLAG-PR70 interacted with both GST-A and GST-Cdc6 but
3A and B, lanes 2–5). Although it did not increase the amount of
not GST alone (Fig. 2C, lanes 1–2 and 6 –7). Compared with
PR70 or the EF1 mutant associated with Cdc6, calcium did
lysates prepared with standard buffer or EGTA, the addition of
increase the association of the A- and C-subunits with GST-
calcium enhanced the binding of PR70 to GST-A, but not to
Cdc6 (Fig. 3B, lanes 2–5). Mutation of EF2 or mutation of both
GST-Cdc6 (Fig. 2C, lanes 4 and 9). Although calcium did not
EF1 and EF2 resulted in loss of the calcium-enhanced binding
enhance the binding of PR70 to GST-Cdc6, it did cause a sig-
of PR70 to GST-A (Fig. 3A, lanes 6 –9) and the calcium-depend-
nificant increase in the amount of A- and C-subunits associated
ent association of the A- and C-subunits with GST-Cdc6 (Fig.
with GST-Cdc6 (Fig. 2C, lanes 9 and 10). Although an excess of
3B, lanes 6 –9).
calcium was used in the experiments shown in Fig. 2C, other
The effect of calcium on the interaction of PR70 with PP2A
experiments showed that enhanced binding of the AC core
was also assessed by expression and immunoprecipitation in
dimer was also observed at calcium concentrations of 100 M
COS-7 cells. Both wild-type PR70 and the EF1 mutants inter-
(not shown).
acted with endogenous PP2A (Fig. 3C, lanes 2– 4). The EF1(-z)
To test the function of the individual EF-hand motifs in the
mutant interacted as well as wild-type PR70, but interaction of
calcium-enhanced interaction with PP2A, GST pulldown
the EF1(x,y) mutant was reduced suggesting that mutation of
experiments were performed with the calcium-binding
the x and y residues causes a structural defect in PR70. Muta-
mutants. Compared with assays in the presence of EGTA, the
tion of EF2, or both EF1 and EF2, resulted in a nearly complete
16108 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 283 • NUMBER 23 • JUNE 6, 2008
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html
PR70 Targets PP2A to Cdc6
GST PR70
A
EF1(-z)
EF2(-z) EF1/2(-z)
A
PR70
PR70
N
E Ca
E Ca
E
Ca
E
Ca
unique
Conserved R3 domain unique
FL-PR70
GST-A
C
pulldown
FYF motif
EF1 EF2
B
PR70
1-575
∆
FL-PR70
N1
125-575
GST-Cdc6
∆N2
136-575
A
pulldown
∆N3
162-575
C
∆C
1-441
1
2
3
4
5
6
7
8
9
y
)
C
B
Vec
c
p
e
y)
y)
V
Em
∆
C
∆
N3
∆
N2
∆
N1
PR70
p
Downloaded from
Em
PR70
EF1(x,
EF1(-z)
EF2(x,
EF2(-z)
EF1/EF2 (x,
EF1/EF2(-z)
FL-PR70
FLAG
Anti-FLAG
A
IP
C
Anti-FLAG
A
IP
A OE
C
C OE
www.jbc.org
FIGURE 4. Mapping of PP2A binding domains in PR70. A, a schematic dia-
1
2
3
4
5
6
7
8
gram of PR70 showing the region containing the conserved R3 domain and
FIGURE 3. The calcium-enhanced association of PP2A with PR70 requires
PR70-unique regions. The truncation mutants used in binding assays are
EF2. FLAG-PR70 and the indicated EF-hand mutants were transiently
shown below with their corresponding designations on the left and amino
expressed in COS-7 cells, and the cells were lysed in standard lysis buffer (N) or
acid numbers on the right. The conserved FYF (FYF motif) and EF-hand motifs
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
lysis buffer containing EGTA (E) or CaCl (Ca). A, GST pulldown assays were
(EF1 and EF2) are also depicted. B, interaction of PR70 truncation mutants with
2
performed with the different lysates using immobilized GST-A. Bound pro-
the A- and C-subunits of PP2A. FLAG-tagged PR70 (PR70) and the indicated
teins were detected by immunoblotting with anti-FLAG (FL-PR70), anti-A-sub-
truncation mutants were transiently expressed in COS-7 cells. The cells were
unit (A), and anti-C-subunit (C) antibodies. B, GST pulldown assays were per-
lysed and the FLAG-tagged proteins immunoprecipitated with anti-FLAG
formed using immobilized GST-Cdc6 as described for A. Lane 1 of panels A and
antibody (Anti-FLAG IP). The immunoprecipitates were resolved by SDS-PAGE
B shows a control pulldown assay using GST alone. C, COS-7 cells were tran-
and immunoblotted with anti-FLAG (FLAG), anti-A-subunit (A), and anti-C-
siently transfected with FLAG-tagged wild-type PR70 or the indicated EF-
subunit (C) antibodies. A control immunoprecipitate using lysate from cells
hand mutants, and lysates prepared with standard buffer were immunopre-
transfected with the empty expression vector (Emp Vec) is shown in the first
cipitated with anti-FLAG antibody. The immunoprecipitates were resolved by
lane.
SDS-PAGE and immunoblotted as described in A. Overexposures of the anti-
A-subunit (A OE) and anti-C-subunit (C OE) immunoblots are also shown.
C-terminal segment that included the PR70-unique region
(⌬C) had little, if any, effect on the interaction with PP2A. How-
loss of interaction with PP2A (Fig. 3C, lanes 5– 8). A longer
ever, deletions of N-terminal regions of the conserved R3
exposure of the blot showed that a weak interaction of the A-
domain, ⌬N2 and ⌬N3, resulted in proteins that failed to inter-
and C-subunits with the EF2 and EF1/EF2 double mutants
act with PP2A. These data indicated that the region between
could still be detected (Fig. 3C, OE). The combination of intact
amino acids 125 and 136 of PR70 were necessary for interaction
cell data and in vitro binding assays provide evidence that PR70
with the PP2A core dimer.
is a calcium binding protein and that interaction with the core
The sequence between residues 125 and 136 of PR70 con-
dimer of PP2A is enhanced by binding of calcium to the second
tains a hydrophobic motif (FYF) that was conserved in PPP2R3
EF-hand motif. Calcium does not affect interaction of PR70
proteins from humans to flies (Fig. 5A). The role of the FYF
with Cdc6 but increases the association of the PP2A core dimer
motif was tested by mutating these residues to alanines (Fig. 5B)
with Cdc6 in a manner dependent upon binding of calcium to
and determining the effects on interaction with the AC core
the second EF-hand of PR70.
dimer. Mutation of any one of these residues resulted in a sig-
PP2A and Cdc6 Bind to Distinct Regions of PR70—Compari-
nificant loss of interaction with endogenous PP2A (Fig. 5C). A
son of the amino acid sequences of the PPP2R3 regulatory sub-
longer exposure of the immunoblot showed that small amounts
units identified a conserved domain in the central region of
of the A- and C-subunits could be detected in immunoprecipi-
PR70 (supplemental Figs. S1 and S4A). The R3 domain is 66%
tates of each of the mutants (not shown). Although the interac-
identical and 82% conserved between human PR70 (PPP2R3B)
tion of the FYF mutants was severely compromised in intact
and PR72 (PPP2R3A). A series of truncation mutants were con-
cells, these mutants still bound to PP2A when assayed in vitro
structed to identify regions within PR70 that were important
by GST pulldown experiments (not shown). These results indi-
for interaction with PP2A and Cdc6. FLAG-tagged mutants
cate that the FYF motif contributes to the interaction of PR70
were expressed in COS-7 cells and immunoprecipitated with
with the A-subunit.
anti-FLAG antibody. The ability of the mutants to incorporate
The N- and C-terminal truncation mutants of PR70 were also
into endogenous PP2A heterotrimers was determined by
tested for their ability to interact with Cdc6. Pulldown experi-
immunoblotting for associated A- and C-subunits. The ⌬N1
ments with GST-Cdc6 were performed with full-length PR70,
mutant contains a deletion of the entire N-terminal PR70-
the ⌬N3, and the ⌬C mutants in the presence and absence of
unique region and interacted with endogenous PP2A subunits
calcium. As expected, full-length PR70 and the ⌬C mutant
to the same extent as full-length PR70 (Fig. 4B). Deletion of a
interacted with the A-subunit of PP2A, whereas the ⌬N3
JUNE 6, 2008 • VOLUME 283 • NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 16109
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html
PR70 Targets PP2A to Cdc6
mutant did not (Fig. 6A). Furthermore, there was an enhanced
A
Human PR70 IPTFYFPRGRP
interaction of PR70 and the ⌬C mutant in the presence of cal-
Human PR72 IPRFYFGEGLP
cium. As shown previously with full-length PR70 (Fig. 2C), the
Mouse PR59 VPAFYFPCGRP
enhanced binding of PR70 and the ⌬C mutant was accompa-
Xenopus PR70 IPKFYFPKGCP
nied by a decrease in the amount of C-subunit associated with
Drosophila PR72 IPRFYFPHGKP
GST-A (Fig. 6A, lanes 3 and 9). This decrease in associated
C-subunit was not observed with the ⌬N3 mutant, which did
B
FYF motif
EF1 EF2
not bind to GST-A. These observations suggest that the
PR70
decrease in C-subunit in the presence of calcium is due to dis-
placement of endogenous regulatory and catalytic subunits
WT 125-IPTFYFPRGRP-135
from GST-A by excess free PR70.
AYF
IPTAYFPRGRP
Both full-length PR70 and the ⌬N3 mutant bound to GST-
FAF
IPTFAFPRGRP
Cdc6 (Fig. 6B). However, only full-length PR70 was able to
FYA
IPTFYAPRGRP
recruit additional A- and C-subunits in the presence of calcium.
Downloaded from
AYA
IPTAYAPRGRP
In contrast, the ⌬C mutant bound very poorly to Cdc6 in the
AAA
IPTAAAPRGRP
presence or absence of calcium. A low level of the A- and C-sub-
units was pulled down with GST-Cdc6 from lysates expressing
C
Vec
the ⌬N3 or ⌬C mutants (Fig. 6B, lanes 5– 6 and 8 –9). Similar
p
amounts of these subunits were also bound to GST-Cdc6 in
Em
D
N3
PR70
AYF
FAF
FYA
AYA
AAA
www.jbc.org
lysates from non-transfected cells (not shown) suggesting that
FLAG
GST-Cdc6 can interact with endogenous A- and C-subunits in
Anti-FLAG
the absence of expressed PR70 (presumably by binding to
A
IP
endogenous PR70). The amounts of A- and C-subunits bound
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
C
to GST-Cdc6 in experiments with the ⌬N3 or ⌬C mutants were
FIGURE 5. A conserved hydrophobic motif is involved in the interac-
increased in the presence of calcium. This observation provides
tion of PR70 with PP2A. A, an alignment of the N-terminal region of the
additional support for the conclusion that calcium can regulate
conserved R3 domains (residues 125–135 of PR70) of PPP2R3 subunits
from various species. Conserved FYF residues are shown in bold. B, a dia-
the association of PP2A with Cdc6 and shows that a C-terminal
gram showing the residues within the PR70 that were changed in the
region of PR70, which includes the PR70-unique domain, is
mutant forms of PR70 listed on the left. Amino acid substitutions are
necessary for interaction with Cdc6.
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
PR70 Regulates Cdc6 Levels—Because phosphorylation of the
cells were lysed, and tagged proteins were immunoprecipitated with anti-
N-terminal regulatory sites of Cdc6 inhibits degradation, loss of
FLAG antibody (Anti-FLAG IP). The immunoprecipitates were resolved by
SDS-PAGE and immunoblotted with anti-FLAG (FLAG), anti-A-subunit (A),
the phosphatase that dephosphorylates these sites should pro-
and anti-C-subunit (C) antibodies. A control immunoprecipitate using
mote accumulation of Cdc6. Therefore, RNA interference was
lysate from cells transfected with the empty expression vector (Emp Vec) is
used to determine if knockdown of PR70 affected the levels of
shown in the first lane.
Cdc6. Knocking down the catalytic subunit of PP2A increased
the levels of endogenous Cdc6 in HeLa cells (Fig. 7A). Treat-
ment of cells with either a control siRNA or an siRNA that
PR70
∆N3
∆C
A
knocks down protein phosphatase 5 had no effect on Cdc6 lev-
els. Knocking down the PR70 subunit also caused a substantial
GST E
Ca GST E
Ca GST E
Ca
increase in the levels of Cdc6 (Fig. 7B). The increase in Cdc6
levels occurred with two PR70 siRNAs targeted to distinct
FLAG
GST-A
regions of the mRNA. The accumulation of Cdc6 following
pulldown
knockdown with PR70-1 siRNA appeared to be greater than
that with PR70-2 siRNA, which is consistent with the greater
C
efficiency of the PR70-1 siRNA in reducing PR70 levels (supple-
B
mental Fig. S2). Phosphorylation site mutants of Cdc6 were
then used to test the role of phosphorylation in the accumula-
FLAG
GST-Cdc6
tion of Cdc6 caused by knockdown of PR70. Knockdown of
pulldown
-A
PR70 caused an increase in the levels of expressed wild-type
C
GFP-Cdc6 compared with transfection with a control siRNA
1
2
3
4
5
6
7
8
9
(Fig. 7B). Transfection with a mutant of Cdc6 in which all three
N-terminal phosphorylation sites had been mutated to phos-
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
pho-mimicking aspartic acid residues (DDD-Cdc6) resulted in
mutant (lanes 7–9) were transiently expressed in COS-7 cells, and the cells
substantially higher levels of expression than those observed
were lysed in the presence of EGTA (E) or CaCl (Ca). GST pulldown assays
2
were performed using immobilized GST-A (panel A) or GST-Cdc6 (panel B),
with the wild-type protein as previously reported (15). PR70-1
and bound proteins were detected by immunoblotting with anti-FLAG
siRNA had little or no effect on the levels of DDD-Cdc6. The
(FLAG), anti-A-subunit (A) or anti-C-subunit (C) antibodies. Control pull-
ability of PR70 knockdown to cause accumulation of Cdc6 was
downs with GST alone (GST) were carried out with all three expressed
proteins using lysates prepared with standard buffer (lanes 1, 4, and 7).
also greatly diminished when the phosphorylation sites were
16110 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 283 • NUMBER 23 • JUNE 6, 2008
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html
PR70 Targets PP2A to Cdc6
A
A
siRNA
Mock
Luc
PP2A-C
PP5
∆N3 ∆C
tor
Cdc6
Vec
pty
Em
Cdc6 Cdc6 + CDK2Cdc6 + PR70
Cdc6 + PR70
Cdc6 + PR70EF1/2
Cdc6 + PR70
PP2A-C
GFP-Cdc6
PR70
Endo-Cdc6
PP5
FLAG-PR70
Downloaded from
B
siRNA
Mock
Luc
PR70-1
PR70-2
Myc-CDK2
Cdc6
Actin
www.jbc.org
PR70
0
B
70
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
PR70
Actin
or
ect
6
6 +
6
6 + PR7
6
6 + PR
C
pty V
plasmid
EV
wtCdc6 DDD-Cdc6 AAA-Cdc6
Em
wtCdc wtCdc
AAA-Cdc
AAA-Cdc
DDD-Cdc
DDD-Cdc
GFP-Cdc6
siRNA
Luc
PR70
Luc
PR70
Luc
PR70
Luc
PR70
GFP-Cdc6
Endo-Cdc6
GPDH
FLAG-PR70
D
Actin
plasmid
EV
wtCdc6 DDD-Cdc6 AAA-Cdc6
Luc siRNA
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 siRNA
PR70 (PR70) or the indicated PR70 mutants. Cells were harvested 24 h after
transfection, and lysates were analyzed by immunoblotting with antibodies
FIGURE 7. Knockdown of PR70 increases the levels of Cdc6. A, HeLa cells were
against Cdc6, actin, FLAG, or myc as indicated at the right. The Cdc6 antibod-
mock transfected (Mock) or transfected with control (Luc), PP2A catalytic subunit
ies detected both the expressed Cdc6 (GFP-Cdc6) and endogenous Cdc6
(PP2A-C), or PP5 (PP5) siRNA. Cells were harvested 48 h after transfection and
(Endo-Cdc6). B, U2OS cells were co-transfected with empty vector or plasmids
immunoblotted with anti-Cdc6, PP2A-C, PR70, or PP5 antibodies. B, HeLa cells
expressing FLAG-PR70 and plasmids expressing GFP-tagged versions of wild-
were mock transfected (Mock) or transfected with control (Luc), PR70-1, or PR70-2
type Cdc6 (wtCdc6), or the AAA (AAA-Cdc6), or DDD (DDD-Cdc6) triple phos-
siRNA. Cells were harvested 48 h after transfection and immunoblotted with anti-
phorylation site mutants of Cdc6. Cells were harvested 24 h later, and lysates
Cdc6, PR70, or actin (as a loading control) antibodies. C, HeLa cells were co-trans-
were analyzed by immunoblotting with antibodies against Cdc6, actin, or the
fected with control or PR70-1 siRNA and plasmids encoding GFP-tagged versions
FLAG epitope as indicated at the right.
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
The effects of overexpressing PR70 on Cdc6 levels were also
hours later, the cells were harvested and lysates were analyzed by immunoblot-
ting with antibodies against Cdc6 or glyceraldehyde-3 phosphate dehydrogen-
determined. When HeLa cells were transfected with expression
ase (GPDH) as a loading control. D, duplicate samples of the lysates described in
plasmids containing the CMV promoter, FLAG-tagged PR70
B, from cells co-transfected with Cdc6 and either luciferase control (Luc siRNA) or
was expressed at levels 5- to 10-fold higher than the endoge-
PR70-1 (PR70) siRNAs were immunoblotted with anti-PR70 antibodies to confirm
knockdown of PR70.
nous protein (not shown). Co-expression of CDK2 and Cdc6
caused a substantial increase in Cdc6 levels as reported previ-
mutated to non-phosphorylatable alanine residues (AAA-
ously (15). Expression of wild-type PR70 caused an increase in
Cdc6). Similar results were seen in U2OS cells. These data indi-
the levels of both co-transfected and endogenous Cdc6 (Fig.
cate that knockdown of PR70 results in an increase in the levels
8A). Expression of the ⌬N3 or EF1/2 mutants, which cannot
of endogenous and exogenous Cdc6 that is dependent on the
interact with the AC core dimer but bind to Cdc6, also
presence of phosphorylatable residues at the N-terminal phos-
increased the levels of Cdc6. In contrast, expression of the ⌬C
phorylation sites.
mutant, which binds to the AC core dimer but not to Cdc6, had
JUNE 6, 2008 • VOLUME 283 • NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 16111
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PR70 Targets PP2A to Cdc6
relatively little effect on the levels of exogenous or endogenous
A
Non-transfected
Cdc6. The potential role of phosphorylation in the effects of
500
overexpressed PR70 was tested using the phosphorylation site
G0/G1 52.1
400
mutants of Cdc6. Although co-expression of PR70 caused some
increase in the levels of the AAA mutant of Cdc6, the effect was
300
S 22.2
much less than its effect on wild-type Cdc6 (Fig. 8B). Similarly,
co-expression of PR70 had little effect on the levels of the
200
G2/M 24.4
DDD mutant of Cdc6 even though endogenous Cdc6 was
increased. Thus, the ability of overexpressed PR70 to cause
100
accumulation of the protein was inhibited when the phos-
phorylation sites in Cdc6 were mutated. The effects of PR70
0
overexpression to cause accumulation of Cdc6 suggest it acts
B
Luc control
in a dominant-negative manner to block Cdc6 dephospho-
400
G0/G1 55.3
rylation (see “Discussion”).
Downloaded from
Knockdown of PR70 Causes G Arrest—The potential role of
1
300
S 22.2
PR70 in progression through G was determined by determin-
1
ing the cell cycle distribution of cells in which PR70 was
200
G2/M 20.9
depleted by RNA interference. Knockdown of PR70 caused
accumulation of cells in G /G and depletion of cells in S and
0
1
www.jbc.org
100
G /M (Fig. 9). The apparent G arrest occurred with either of
2
1
two siRNAs that target distinct regions of PR70. The level of G1
0
arrest correlated with the extent of PR70 knockdown. The
C
PR70 siRNA-2
lower levels of PR70 achieved with the PR70 siRNA-1 com-
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
No. of Cells
pared with PR70 siRNA-2 corresponded to a greater increase in
600
G0/G1 65
the number of G cells (76% versus 65%). The G arrest caused
1
1
S 16.3
by knockdown of PR70 supports a role for this PP2A regulatory
400
subunit in progression through G phase.
1
G2/M 17.9
DISCUSSION
200
The formation of pre-replicative complexes during the initi-
ation of DNA replication is regulated, in part, by the availability
of Cdc6. Cyclin-dependent kinases phosphorylate regulatory
0
800
D
PR70 siRNA-1
sites within the N-terminal domain of Cdc6 and block ubiquiti-
nation by APC/Ccdh1 and subsequent degradation by the pro-
G0/G1 76.5
teasome (15). The results reported here help establish the form
600
of PP2A complexed with the PR70 regulatory subunit as a phys-
S 12.1
iological Cdc6 phosphatase and are consistent with a model in
400
which PR70 targets PP2A to Cdc6 through direct protein-pro-
G2/M 10.7
tein interactions. Knockdown of PR70 by RNA interference
200
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-nega-
0
tive manner to also increase the levels of Cdc6. The observa-
0
200
400
600
800
tions that increased protein levels did not occur with phospho-
FL3 Area
rylation site mutants of Cdc6 are consistent with a role for PR70
in regulating Cdc6 phosphorylation and stability. A novel
E
aspect of this model is the potential regulation of Cdc6 dephos-
phorylation 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 intracel-
Non-transfected
Luc controlPR70 siRNA-2
PR70 siRNA-1
lular calcium can regulate the accumulation of Cdc6 and initi-
PR70
ation of DNA replication. However, it remains to be deter-
mined if physiological changes in intracellular calcium
GPDH
analyzed by flow cytometry. The data are plotted as the number of cells versus
FIGURE 9. Knockdown of PR70 causes G arrest. A–D, U2OS cells were left
DNA content determined by FL3 area. The percentages of cells in G /G , S, and
1
0
1
untreated (A), or transfected with control siRNA (B), PR70-2 siRNA (C), or
G /M phases are indicated. E, duplicate transfections were harvested after
2
PR70-1 siRNA (D). Forty-eight hours later, the cells were harvested and
48 h and immunoblotted with anti-PR70 antibody to confirm knockdown.
16112 JOURNAL OF BIOLOGICAL CHEMISTRY
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Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html
PR70 Targets PP2A to Cdc6
concentrations are sufficient to regulate association of PP2A
previous study showed that increases in phosphorylation and
with Cdc6.
stability of Cdc6 enhance formation of pre-replicative com-
Biochemical analysis of the interaction of PR70 with the AC
plexes (15). The increase in pre-replicative complex formation
core dimer suggested an unanticipated mechanism for regulat-
would be expected to enhance entry into S phase. Consistent
ing PP2A activity. Experiments with N-terminal truncation
with this idea, expression of exogenous wild-type Cdc6 leads to
mutants showed that PR70 can associate with Cdc6 independ-
accelerated entry into S phase (14). However, exogenous
ently of the A- and C-subunits. This observation contrasts with
expression of a non-phosphorylatable (AAA) mutant of Cdc6
the prevailing view of PP2A in which the regulatory subunits
(5) or an N-terminally truncated version of Cdc6, missing the
have been thought to be constitutively associated with the core
CDK phosphorylation sites and destruction motifs recognized
dimer (18 –20). The importance of the existence of PP2A in
by APC/Ccdh1 (14), inhibit initiation of DNA replication and
heterotrimeric forms is supported by data showing that some
entry into S phase. It is possible that, even though phosphoryl-
PP2A regulatory subunits are only stable when incorporated
ation is required for stabilization of Cdc6 and assembly of pre-
into holoenzymes (29 –31). Overexpression of the PPP2R2 fam-
replicative complexes during G , an additional Cdc6 dephos-
1
ily member, B␥, leads to proteasome-dependent degradation of
phorylation or degradation step is needed to initiate DNA
Downloaded from
the free protein but not protein incorporation into holoen-
replication. Knockdown or overexpression of PR70 might
zymes (31). In contrast, several lines of evidence indicate that
inhibit this step and retard entry into S phase. Although the
members of the PPP2R3/PR72 family are stable regardless of
ability of PR70 knockdown to cause G arrest is consistent with
1
whether or not they are incorporated into holoenzymes. As
regulation of Cdc6, an equally likely possibility is that PR70
shown here, mutants of PR70 that cannot bind to PP2A accu-
plays other roles during G . PR70 may regulate the activity of
1
www.jbc.org
mulate to the same levels as the wild-type protein. The apparent
other proteins involved in cell cycle control, either through
stability of PR70 is also independent of interaction with Cdc6,
PP2A-mediated dephosphorylation or actions that are inde-
because a mutant (⌬C) that interacts poorly with Cdc6 accu-
pendent of PP2A.
mulates to similar levels. Similarly, mutations of the related
The effects of calcium on the PR70-dependent association of
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
PR72 subunit that block interaction with the AC core dimer
PP2A with Cdc6 are consistent with a more general role for the
have no effect on the levels of expressed protein (27). In addi-
PPP2R3/PR72 family in mediating calcium-regulated dephos-
tion, the PR59 subunit is not degraded following loss of the
phorylation. All four members of this family contain conserved
A-subunit (31). Thus, in contrast to the PPP2R2/B and
EF-hand sequence motifs (supplemental Fig. S1). The EF-hands
PPP2R5/B56 families of PP2A regulatory subunits, members of
of PR72 are also functional calcium binding sites, and, similar to
the PPP2R3/PR72 family are stable proteins whose levels and
PR70, calcium binding to the second EF-hand enhances inter-
functional interactions with substrates and other proteins may
action with the A-subunit (27). PR72 has been shown to medi-
be independent of the core dimer of PP2A.
ate calcium-dependent dephosphorylation of threonine-75 in
The stability of expressed PR70 may also account for its abil-
the dopamine- and cAMP-regulated phosphoprotein of 32 kDa
ity to act in an apparent dominant-negative manner to increase
(DARPP-32). This report showed that, in addition to the role of
the levels of Cdc6. Excess free PR70 would associate with Cdc6
EF-hand 2 in interaction with the AC core dimer, calcium bind-
and displace endogenous PR70-AC holoenzyme. Loss of the
ing to EF-hand 1 increased the phosphatase activity of the
active AC core dimer from Cdc6 would inhibit dephosphoryl-
PR72-holoenzyme toward DARPP-32 (32). Both PR72 and its
ation leading to decreased ubiquitination by APC/Ccdh1 and
alternative splice variant (PR130) have been reported to inter-
increase protein levels. A dominant-negative action of overex-
act with the mammalian Naked cuticle protein and regulate
pressed PR70 is supported by observations that the effects on
Wnt signaling (33, 34). Calcium may therefore influence Wnt
Cdc6 levels are not dependent on interaction of PR70 with the
signaling through recruitment and/or regulation of PP2A asso-
AC core dimer (e.g. the ⌬N3 and EF1/2 mutants) but are
ciated with Naked cuticle. The other member of the PPP2R3
dependent on interaction with Cdc6 (e.g. the ⌬C mutant). Like
family, PR59, targets PP2A to the retinoblastoma-related p107
knockdown of PR70, the dominant-negative actions of PR70 to
protein (35) and may provide a mechanism for calcium regula-
increase Cdc6 levels appear to be dependent on intact phospho-
tion of the cell cycle functions of p107.
rylation sites, because the levels of co-expressed DDD and AAA
The sites involved in the interaction with PP2A and Cdc6
mutants of Cdc6 were not significantly affected. Forced over-
mapped to distinct regions of PR70, consistent with a role in
expression of the related PR72 subunit has also been reported
bridging the two proteins. The N-terminal domain of PR70,
to act in a dominant-negative manner. Expression of either
which is not conserved with other members of the PPP2R3
wild-type PR72 or an EF-hand 2 mutant, which cannot bind the
family, is not required for interaction with either PP2A or Cdc6.
AC core dimer, both cause G arrest in U2OS cells (27).
Deletion of the C-terminal region, including the PR70-unique
1
The accumulation of cells with G /G DNA content follow-
sequence and a portion of the conserved R3 domain, had no
0
1
ing knockdown by RNA interference is consistent with an
effect on interaction with the PP2A core dimer but severely
important role for PR70 in progression of cells through into S
inhibited binding to Cdc6. Conversely, deletion of N-terminal
phase. Similarly, overexpression of a fragment that contains the
sequences within the conserved R3 domain blocked binding to
complete R3 domain and the C terminus of PR70 (termed PR48
the A-subunit but had no effect on interaction with Cdc6. The
or ⌬N1 in this study) also causes G arrest, presumably through
N-terminal region required for interaction with the A-subunit
1
a dominant-negative action (17). The G arrest in cells depleted
contains an FYF amino acid motif that plays a role in the inter-
1
of PR70 coincides with an increase in Cdc6 protein levels. A
action and is conserved between members of the PPP2R3 fam-
JUNE 6, 2008 • VOLUME 283 • NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 16113
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/04/09/M710313200.DC1.html
PR70 Targets PP2A to Cdc6
ily. The A-subunit of PP2A is a HEAT repeat protein (36). The
10. Coverley, D., Pelizon, C., Trewick, S., and Laskey, R. A. (2000) J. Cell Sci.
FYF motif in PR70 resembles the FG amino acid repeats (FXFG
113, 1929 –1938
and GLFG) within the nucleoporin family of nuclear pore pro-
11. Mendez, J., and Stillman, B. (2000) Mol. Cell Biol. 20, 8602– 8612
12. Alexandrow, M. G., and Hamlin, J. L. (2004) Mol. Cell Biol. 24, 1614 –1627
teins. The nucleoporins interact with nuclear transport factors,
13. Harper, J. W., Burton, J. L., and Solomon, M. J. (2002) Genes Dev. 16,
including importin-, which are also HEAT repeat proteins.
2179 –2206
The FG repeats of nucleoporins bind to shallow hydrophobic
14. Petersen, B. O., Wagener, C., Marinoni, F., Kramer, E. R., Melixetian, M.,
pockets in importin- (37, 38). The A-subunit of PP2A contains
Denchi, E. L., Gieffers, C., Matteucci, C., Peters, J. M., and Helin, K. (2000)
exposed hydrophobic surfaces, predicted to play a role in inter-
Genes Dev. 14, 2330 –2343
action with the regulatory subunits (36), that are possible sites
15. Mailand, N., and Diffley, J. F. (2005) Cell 122, 915–926
16. Duursma, A., and Agami, R. (2005) Mol. Cell Biol. 25, 6937– 6947
of interaction with the FYF motif of PR70.
17. Yan, Z., Fedorov, S. A., Mumby, M. C., and Williams, R. S. (2000) Mol. Cell
The requirement for the N-terminal region of the R3 domain
Biol. 20, 1021–1029
of PR70 for binding to the A-subunit is distinct from results
18. Virshup, D. M. (2000) Curr. Opin. Cell Biol. 12, 180 –185
observed with the PR72 protein. A fragment of PR72 consisting
19. Janssens, V., and Goris, J. (2001) Biochem. J. 353, 417– 439
of amino acids 219 – 473 (corresponding to residues 257–509 of
20. Silverstein, A. M., Davis, A. J., Bielinski, V. A., Esplin, E. D., Mahmood,
Downloaded from
PR70) interacts with the A-subunit in the yeast two-hybrid
N. A., and Mumby, M. C. (2003) in Handbook of Cellular Signaling (Brad-
assay (27). This fragment of PR72 is missing the N-terminal
shaw, R. A., and Dennis, E. A., eds) pp. 405– 415, Academic Press, San
Diego, CA
region of the R3 domain. Two fragments of PR72 containing
21. Stevens, I., Janssens, V., Martens, E., Dilworth, S., Goris, J., and Van Hoof,
putative A-subunit binding domains prepared by in vitro trans-
C. (2003) Eur. J. Biochem. 270, 376 –387
lation (corresponding to residues 234 –339 and 378 – 436 of
22. Pearson, G. W., Earnest, S., and Cobb, M. H. (2006) Mol. Cell Biol. 26,
www.jbc.org
PR70) interacted with the A-subunit in vitro using GST pull-
3039 –3047
down assays (39). These PR72 fragments also do not contain the
23. Ali, A., Zhang, J., Bao, S., Liu, I., Otterness, D., Dean, N. M., Abraham,
conserved N-terminal region of the R3 domain that was neces-
R. T., and Wang, X. F. (2004) Genes Dev. 18, 249 –254
24. Mumby, M. C., Green, D. D., and Russell, K. R. (1985) J. Biol. Chem. 260,
sary for interaction of PR70 with the AC core dimer in our
at Centro Nacional de Investigaciones Oncológicas, on May 28, 2010
13763–13770
assays. These observations indicate that additional regions of
25. Maruyama, K., Mikawa, T., and Ebashi, S. (1984) J. Biochem. (Tokyo) 95,
PR70, beyond those required in PR72, are required for binding
511–519
to the A-subunit, or that the differences observed are due to
26. Sontag, E., Fedorov, S., Kamibayashi, C., Robbins, D., Cobb, M., and
different assays employed.
Mumby, M. (1993) Cell 75, 887– 897
In summary, the present study shows that the PR70 regula-
27. Janssens, V., Jordens, J., Stevens, I., Van Hoof, C., Martens, E., De Smedt,
tory subunit targets PP2A to Cdc6 and that PP2A is likely to be
H., Engelborghs, Y., Waelkens, E., and Goris, J. (2003) J. Biol. Chem. 278,
10697–10706
a physiological Cdc6 phosphatase. The targeting of PP2A to
28. Strynadka, N. C., and James, M. N. (1989) Annu. Rev. Biochem. 58,
Cdc6 is enhanced by binding of calcium to PR70 raising the
951–998
possibility that changes in intracellular calcium can influence
29. Silverstein, A. M., Barrow, C. A., Davis, A. J., and Mumby, M. C. (2002)
formation of pre-replicative complexes through regulation of
Proc. Natl. Acad. Sci. U. S. A. 99, 4221– 4226
Cdc6 dephosphorylation.
30. Li, X., Scuderi, A., Letsou, A., and Virshup, D. M. (2002) Mol. Cell Biol. 22,
3674 –3684
REFERENCES
31. Strack, S., Ruediger, R., Walter, G., Dagda, R. K., Barwacz, C. A., and
1. Bell, S. P., and Dutta, A. (2002) Annu. Rev. Biochem. 71, 333–374
Cribbs, J. T. (2002) J. Biol. Chem. 277, 20750 –20755
2. Diffley, J. F. (2004) Curr. Biol. 14, R778 –R786
32. Ahn, J. H., Sung, J. Y., McAvoy, T., Nishi, A., Janssens, V., Goris, J., Green-
3. Williams, R. S., Shohet, R. V., and Stillman, B. (1997) Proc. Natl. Acad. Sci.
gard, P., and Nairn, A. C. (2007) Proc. Natl. Acad. Sci. U. S. A. 104,
U. S. A. 94, 142–147
9876 –9881
4. Yan, Z., DeGregori, J., Shohet, R., Leone, G., Stillman, B., Nevins, J. R., and
33. Creyghton, M. P., Roel, G., Eichhorn, P. J., Hijmans, E. M., Maurer, I.,
Williams, R. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3603–3608
Destree, O., and Bernards, R. (2005) Genes Dev. 19, 376 –386
5. Jiang, W., Wells, N. J., and Hunter, T. (1999) Proc. Natl. Acad. Sci. U. S. A.
34. Creyghton, M. P., Roel, G., Eichhorn, P. J., Vredeveld, L. C., Destree, O.,
96, 6193– 6198
and Bernards, R. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 5397–5402
6. Herbig, U., Griffith, J. W., and Fanning, E. (2000) Mol. Biol. Cell 11,
35. Voorhoeve, P. M., Hijmans, E. M., and Bernards, R. (1999) Oncogene 18,
4117– 4130
515–524
7. Petersen, B. O., Lukas, J., Sorensen, C. S., Bartek, J., and Helin, K. (1999)
36. Groves, M. R., Hanlon, N., Turowski, P., Hemmings, B. A., and Barford, D.
EMBO J. 18, 396 – 410
(1999) Cell 96, 99 –110
8. Pelizon, C., Madine, M. A., Romanowski, P., and Laskey, R. A. (2000)
37. Bayliss, R., Littlewood, T., and Stewart, M. (2000) Cell 102, 99 –108
Genes Dev. 14, 2526 –2533
38. Bayliss, R., Littlewood, T., Strawn, L. A., Wente, S. R., and Stewart, M.
9. Delmolino, L. M., Saha, P., and Dutta, A. (2001) J. Biol. Chem. 276,
(2002) J. Biol. Chem. 277, 50597–50606
26947–26954
39. Li, X., and Virshup, D. M. (2002) Eur. J. Biochem. 269, 546 –552
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