Page 3

6.8, 5 m

MgCl

, and 60 m

KCl) for 1 h at room temperature.

The membrane was then incubated in IMK buffer containing 5
␮Ci/ml

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 intro-

duced into the EF-hands of PR70 as described in the manual for
the QuikChange Multi Site-directed mutagenesis kit (Strat-
agene) 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

Ј-

CAAGTTCTGGGAGCTGGCCACGGCCCACGACCTGCTC-
ATCG-3

Ј (sense strand) and 5Ј-CGATGAGCAGGTCGTGGG-

CCGTGGCCAGCTCCCAGAACTTG-3

Ј (antisense strand),

EF1(-z) 5

Ј-TTGTGCCGCGCCAGGTTGTCCGCGTCGATGA-

GCGCTCATCGACGCGGACAACCTGGCGCGGCACAA-3

(sense strand) and 5

Ј 3 3Ј (antisense strand), EF2(x,y) 5Ј-TGGT-

TCCGCTGCATGGCCCTGGCCGGGGACGGCGCCCTG-3

(sense strand) and 5

Ј-CAGGGCGCCGTCCCCGGCCAGGGC-

CATGCAGCGGAACCA-3

Ј (antisense strand), and EF2(-z), 5Ј-

GCGCCCTGTCCATGTTCCAGCTCGAGTACTTCTAC-3

(sense strand) and 5

Ј-GTAGAAGTACTCGAGCTGGAACAT-

GGACAGGGCGC-3

Ј (antisense strand). Mutations in both EF-

hands were introduced using the EF1 mutant cDNAs as tem-
plate for PCR with primers for introduction of EF2 point
mutants. All mutations were verified by automated sequencing.

PR70 truncation mutants were generated by PCR amplifi-

cation 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) 5Ј-CGGGATCCGCCACCATG-

GATGACATG-3

Ј, ⌬N3 (aa 162–575) 5Ј-CGGGATCCAG-

GACTCCGTCAACGTG-3

Ј, and ⌬C (aa 1– 441) 5Ј-CCCA-

AGCTTCATCTGGCAGAGGCAGTC-3

Ј.

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 under-
lined): AYF, 5

Ј-GCCAAAGCATTCCGACCGCCTACTTCCC-

CAGAGGACG-3

Ј (sense strand) and 5Ј-CGTCCTCTGGGGA-

AGTAGGCGGTCGGAATGCTTTGGC-3

Ј (antisense strand),

FAF, 5

Ј-CCAAAGCATTCCGACCTTCGCCTTCCCCAGAG-

GACGCC-3

Ј (sense strand) and 5Ј-GGCGTCCTCTGGGGAA-

GGCGAAGGTCGGAATGCTTTGG-3

Ј (antisense strand),

FYA, 5

Ј-GCATTCCGACCTTCTACGCCCCCAGAGGACGC-

CCGC-3

Ј (sense strand) and 5Ј-GCTTTCGTCCTCTGGGGGC-

GTAGAAGGTCGGAATGC-3

Ј (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

Ј-CATTCCGACCGCCTACGCCCCCAGAGGACGCCCG-3Ј

(sense strand) and 5

Ј-CGGGCGTCCTCTGGGGGCGTAGGC-

GGTCGGAATG-3

Ј (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

Ј-GCA-

TTCCGACCGCCGCCGCCCCCAGAGGACG-3

(sense

strand) and 5

Ј-CGTCCTCTGGGGGCGGCGGCGGTCGG-

AATGC-3

Ј (antisense strand). All mutations were verified

by automated sequencing.

Cloning of GST-tagged PR70 and EF-hand Mutants

—PR70

and PR70 EF-hand mutant cDNA were cloned into the pGEX-
4T-1 vector (Amersham Biosciences). The cDNAs were ampli-
fied by PCR using the pCMV-Tag2B vector containing the full-
length PR70 or EF-hand mutant cDNA as template with the
following primers: 5

Ј-CGGGATCCATGCCGCCCGGCAA-

AGT-3 (sense strand) and 5

Ј-ATTTGCGGCCGCTCACAG-

CGGCTCCAGGTC-3

Ј (antisense strand). The products were

digested with BamHI and NotI and ligated into pGEX 4T-1,
which had been cut with the same restriction enzymes. The
resulting constructs encode the GST protein fused to the N
terminus of full-length PR70 or EF-hand mutant proteins. The
sequences were verified by automated sequencing.

Expression and Purification of GST-Cdc6, GST-A, and GST-

PR70 Fusion Proteins

—A GST-Cdc6 fusion protein was pre-

pared by a modification of a method previously described (6).
Briefly, 1 liter of Sf9 cells (2

ϫ 10

cells/ml) was infected with

recombinant GST-Cdc6 baculovirus (a gift of Dr. Ellen Fan-
ning, 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 m

Tris-HCl, pH 7.4, 100 m

NaCl, 5 m

KCl,

0.5 m

MgCl

, 0.5% Nonidet P-40, 1 m

dithiothreitol, 10 m

NaF, 1 m

EGTA, 2 m

EDTA, and a protease inhibitor tablet

(Roche Applied Science)) using a Dounce homogenizer. Lysates
were centrifuged at 30,000

ϫ g for 30 min at 4 °C to remove

cellular debris, and the lysate was mixed with 2 ml of glutathi-
one-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

NaCl, and once with PBS containing

1.5 m

NaCl and 0.1% (v/v) Nonidet P-40, and then re-equili-

brated in PBS. The GST-Cdc6 fusion protein immobilized on
glutathione agarose beads was resuspended in buffer B (20 m

HEPES, pH 7.6, 100 m

KCl, 1 m

dithiothreitol, 1 m

EDTA,

and 50% glycerol) and stored at

Ϫ80 °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 gluta-
thione agarose beads were resuspended in buffer B and stored
at

Ϫ80 °C until use.

GST Pulldown Assays

—GST, GST-A, and GST-Cdc6 immo-

bilized 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 m

EDTA or 10 m

CaCl

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 con-
centration of 50

␮

in some experiments. Following incuba-

tion, the sample was washed three times with IP lysis buffer
supplemented with EGTA, CaCl

, or CaCl

and calpeptin, and

the beads were collected by centrifugation. After washing, the
bound proteins were solubilized in 60

␮l of 2ϫ SDS-PAGE

loading buffer. 30

␮l of solubilized protein was resolved on a

10% SDS-PAGE gel and transferred to a nitrocellulose mem-
brane. The membrane was probed with anti-FLAG monoclonal

PR70 Targets PP2A to Cdc6

16106

JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 283 • NUMBER 23 • JUNE 6, 2008

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