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Departments of
*
Pediatrics,
Cardiothoracic Surgery, and
Pathology, Stanford University, Stanford, CA 94305; and
§
Department of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of HLA-DQA03011 (DQ65–79) blocks
cell cycle progression from early G1 to the G1
restriction point, which inhibits cyclin-dependent kinase-2 activity
and phosphorylation of the retinoblastoma protein. A yeast two-hybrid
screen identified proliferating cell nuclear Ag (PCNA) as a cellular
ligand for this peptide, whose interaction with PCNA was further
confirmed by in vitro biochemistry. Electron microscopy demonstrates
that the DQ65–79 peptide enters the cell and colocalizes with PCNA in
the T cell nucleus in vivo. Binding of the DQ65–79 peptide to PCNA did
not block polymerase 
(pol )-dependent DNA replication in vitro.
These findings support a key role for PCNA as a sensor of cell cycle
progression and reveal an unanticipated function for conserved regions
of HLA molecules. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of HLA-DQA03011 (DQ65–79) is profoundly
anti-proliferative, blocking cell cycle progression in G1
and inhibiting cyclin-dependent kinase
(CDK)3-2 activity
(6). In the present study, we used a yeast two-hybrid
screen to identify the cellular ligand for this anti-proliferative
peptide. DQ65–79 binds specifically to proliferating cell nuclear Ag
(PCNA) and blocks downstream events such as the phosphorylation of the
cell cycle checkpoint protein retinoblastoma (Rb). These findings
clarify the mechanism of action of this HLA-derived peptide, suggest
novel functions for HLA molecules, and support the role of PCNA as a
cell cycle "sensor" in mammalian cells. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The yeast two-hybrid screen was performed as described in the MATCHMAKER Two-Hybrid System (Clontech, Palo Alto, CA). Synthetic oligonucleotides encoding residues 65–79 of HLA-DQA03011 were designed using the most frequent codon for each amino acid. The peptide sequences were fused to the carboxyl terminus of the GAL4 DNA-binding domain in plasmid vector pAS1 (a gift from Dr. S. J. Elledge, Baylor College of Medicine), which results in the plasmid pAS-DQ (also named pXL-17). pAS-B2702, which has HLA class I B2702 84–75/75–84 peptide sequences, was constructed similarly as pAS-DQ. Other control plasmids pAS-lamin, pAS-p53, and pAS-CDK2 are gifts from Dr. S. J. Elledge. The plasmid vector, pACT2, was purchased from Clontech. Poly-A mRNA was extracted from human PBL 6 days after PHA-P activation and was reverse transcribed into cDNA. The cDNA expression library was generated by fusing cDNA to the carboxyl terminal of the GAL4 activation domain in the pACT2 plasmid, yielding a complexity >106. The library and pAS-DQ plasmids were cotransformed into the yeast strain Y190 (from Clontech), and screens were performed as described by Clontech matchmaker yeast two-hybrid protocol. "Interacting" clones isolated in the library screen were retransformed with either with pAS-DQ or with other "bait" plasmids. Cotransformation experiments were performed in duplicate, and transformants were plated with synthetic minimal media SD-Trp-Leu-HIS plus 3AT (30 mM 3-amino-1,2,4-triazole, catalogue no. A-8056, Sigma, St. Louis, MO). After incubation at 30°C for 3 days, a ß-galactosidase filter assay was performed. Clones positive with pAS-DQ but not with other "bait" plasmids were further characterized. Library plasmids were isolated from positive clones and sequenced (Sequenase 1.0 DNA Sequencing Kit, United States Biochemical, Cleveland, OH). The DNA sequences generated were analyzed for homology in the GenBank database (http://www.ncbi.nlm.nih.gov).
In vitro binding of PCNA to peptide
Peptides were synthesized and HPLC purified as described (3). Where indicated, a six-histidine-tag (HIS-) was added to the amino terminus. The histidine tag did not affect the anti-proliferative function of DQ65–79 peptide (data not shown). Stock peptide solutions were prepared in DMSO, and exact concentrations were determined by amino acid analysis (PAN Facility, Beckman Center, Stanford University). T cell proliferation was assayed as described (6). The peptide/PCNA interaction in vitro was assayed by ELISA. Microtiter wells were coated with 200 µl of 1 µM recombinant purified PCNA in PBS overnight at 4°C, washed with PBST (PBS plus 0.1% Tween 20), then blocked with 5% nonfat milk in PBST for 2 h at room temperature. Wells were then incubated with 1) peptide for 1 h at room temperature; 2) anti-tetra histidine mAb (Qiagen, Chatsworth, CA) diluted to 1:1000 in PBST for 1 h at room temperature; 3) HRP-conjugated anti-mouse Ab at 1:1000 in PBST for 1 h at room temperature; and 4) o-phenylenediamine (0.5 mg/ml in 0.25 M citrate buffer, pH 5.0, with 0.03% H2O2). Wells were washed extensively with PBST between each step. The reaction was stopped by addition of 30 µl 2 N H2SO4, and OD was determined on a SpectraMax 340 Microplate Reader (Molecular Devices, Sunnyvale, CA) using a 492-nm pore size filter. All experiments were performed in triplicate, and the SD was <10%.
Electron microscopy
PBL were isolated by Ficoll-Hypaque density centrifugation and cultured for 6 days in RPMI 1640 supplemented with 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 µg/ml PHA-P. On day 6, 5 x 106 PBL cells were incubated with either 40 µM HIS-DQ or the equivalent amount of DMSO for 1 h and then fixed with 5% glutaraldehyde in PBS (Sigma) for 1 h at room temperature. The cells were washed in several changes of PBS, dehydrated in a series of ethanol treatments, and embedded in LR White (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were blocked in PBS containing 5% BSA and 0.5% goat serum for 1 h at room temperature. PCNA was labeled by sheep anti-human PCNA (The Binding Site, Birmingham, U.K.), rabbit anti-sheep IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), and goat anti-rabbit-10 nm colloidal gold (AutoProbe EM GAR G10, Amersham Life Sciences, Arlington Heights, IL). HIS-DQ65–79 was labeled by anti-penta HIS (BSA free, mouse IgG1 mAb, Qiagen) and goat anti-mouse IgG-30 nm colloidal gold (AutoProbe EM GAR G30, Amersham Life Sciences). PCNA and HIS-DQ65–79 were applied in two separate rounds of staining. Briefly, each round consisted of the application of a primary Ab/blocking buffer to the sections for overnight incubation at 4°C, then incubation with the appropriate secondary Ab in blocking buffer and/or colloidal gold solution, each for 3 h at room temperature. The sections were then washed in distilled water and lightly counterstained with uranyl acetate and lead citrate. The sections were viewed with a Hitachi H-300 electron microscope (Hitachi, Tokyo, Japan).
Elongation of labeled singly primed DNA by polymerase (pol
) holoenzyme
The assay was conducted essentially as described
(7). Briefly, the reaction mixture (20 µl) contained 20
mM HEPES, pH 7.5, 0.4 mM DTT, 150 µg/ml BSA, 6 mM MgAc, 100 µM
dNTPs (dCTP, dGTP, dTTP), 1 mM ATP, 15 µM
[-32P]dATP, 8.7 fmol singly primed M13, 0.5
µg human single-stranded binding protein (replication protein A), 50
fmol replication factor C, 0.25 U pol , 43 pmol peptide, and PCNA as
indicated. After a 30-min incubation at 37°C, the reaction mixture
was adjusted to 10 mM EDTA, loading dye was added, and samples were
electrophoresed on 1.8% alkaline gel. The gel was dried and
autoradiographed.
Rb phosphorylation
IL-2-dependent T cells were activated with 100 U/ml IL-2 in the presence of DQ65–79 (40 µM), DQ72D (40 µM), rapamycin (100 nM), or DMSO alone (media control) at time 0. Cells (5 x 106) were harvested either at 24 or 48 h after treatment, and whole cell lysates were prepared and run on a 10% SDS-PAGE, transferred to nitrocellulose, and probed with Abs to Rb (Santa Cruz Biotechnology, Santa Cruz, CA) and HSC70 (loading control).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A yeast two-hybrid screen (8) was used to identify
potential ligands for the anti-proliferative DQ65–79 peptide. The
results of screening 
2 x 107
transformants are summarized in Table I
.
Of the 18 clones that were positive in multiple screens, only one, a
clone encoding residues 178–261 of PCNA, interacted specifically with
DQ65–79. When other proteins including CDK2, lamin, p53, and a
similar-sized peptide corresponding to residues 75–84 of HLA-B2702
were used a "bait," no interaction with PCNA could be demonstrated
(not shown).
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,
A and B), demonstrating that the DQ65–79/PCNA
interaction was direct and did not require intermediate or additional
factors. To define the residues within the DQ65–79 peptide critical
for PCNA binding, a panel of synthetic peptides was prepared in which a
six-histidine tag was added to the amino terminus for detection and
each residue was individually substituted with serine (Fig. 1C). Substitution at a cluster of three hydrophobic residues
(isoleucine at position 75, valine at 76, and isoleucine at 77)
prevented the binding of the DQ65–79 peptide to PCNA. With the
exception of the HIS-73S peptide, there was a strong correlation
between the binding of each peptide to PCNA and the ability of that
peptide to inhibit T cell proliferation.
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Characterization of the interaction of PCNA and the DQ65–79 in
vitro suggests that the anti-proliferative effect of the DQ65–79
peptide may be due to its binding of PCNA in vivo. If the binding of
the DQ65–79 peptide to PCNA blocks cell cycle progression, the
DQ65–79 peptide should enter the cell and colocalize with PCNA. To
test this hypothesis, electron microscopy experiments were performed.
As shown in Fig. 2, in HIS-DQ65–79
peptide-treated T cells, HIS-DQ65–79 (labeled by 30 nm gold particles)
was found almost exclusively in the nucleus and in very close proximity
to PCNA (labeled by 10-nm gold particles) in numerous instances per
grid. When both primary Abs were omitted, no gold particles were
detected, demonstrating that the secondary Abs specifically recognize
the primary Abs rather than nonspecific epitopes in other proteins. In
DMSO-treated T cells, PCNA distribution was essentially identical with
that of the samples treated with the HIS-DQ65–79 peptide. These
findings clearly demonstrate that DQ65–79 crosses the cell plasma
membrane and interacts physically with PCNA in vivo in the T cell
nucleus.
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-dependent DNA
replication in vitro
PCNA plays important roles in DNA replication, DNA repair, and
regulation of the cell cycle. PCNA interacts with DNA pol and
replication factor C to form a processive DNA polymerase complex
(9, 10, 11). Synthetic peptides corresponding to p21, a cell
cycle inhibitor, bind PCNA and block the function of PCNA in the DNA
polymerase complex in vitro (12). To determine whether
DQ65–79 affects DNA replication by direct binding to PCNA, peptides
were added to an in vitro system that measures pol -dependent DNA
synthesis (7). None of the peptides tested, DQ65–79,
DQ74S, and DQ72D, had an effect on pol -dependent DNA synthesis
(Fig. 3), demonstrating that the
anti-proliferative effect of DQ65–79 does not involve effects on
DNA pol replication per se.
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To evaluate the effects of the DQ65–79 peptide on the G1
check protein Rb, activated T cells were treated either with the
inhibitory DQ65–79 peptide or with a peptide in which glutamine was
substituted for aspartic acid at residue 72 (DQ72D). This substituted
peptide does not inhibit T cell proliferation (6) and does
not bind to PCNA in vitro (data not shown). Rb was normally
phosphorylated in cells incubated with DQ72D, but was not
phosphorylated in DQ65–79-treated cells (Fig. 4). Both rapamycin and DQ65–79 block
cell cycle progression in a similar fashion (6), and we
found that Rb was hypophosphorylated in cells treated with either
DQ65–79 or rapamycin (Fig. 4), underscoring the mechanistic similarity
between these two immunosuppressive agents. The Rb phosphorylation
pattern also suggests that DQ65–79 and rapamycin both affect some cell
cycle control point upstream of the Rb in the early G1 to
G1 restriction point regulation pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic peptides derived from p21 interact with PCNA to block
replication in vitro but have no effect on DNA synthesis in intact
cells (12), most likely because they are not able to
penetrate the cell membrane. In contrast, the DQ peptide interacts with
PCNA in T cell nuclei when added to cells in culture and is highly
anti-proliferative (6). This may be related to the
fact that the p21 peptide has no obvious secondary structure (Ref.
12 and this study; circular dichroism data not shown),
while the DQ peptide has a strongly helical profile (data not
shown). In addition, although both peptides can interact with PCNA,
their mechanisms of inhibitory action are different as the p21 peptide
binds to PCNA to block pol -dependent replication (7, 12) while the DQ65–79 peptide does not.
PCNA forms a quaternary complex with CDK, cyclin D, and p21, suggesting that PCNA may play an important role in cell cycle regulation beyond its function in the DNA replication machinery (13, 14, 15). CDKs regulate progression through the cell cycle. Activation of CDKs is a complex process, modulated by both positive and negative regulatory signals. These CDKs are positively regulated through association with cyclin molecules and by threonine phosphorylation and negatively regulated through association with kinase inhibitors from the Ink, Cip, or Kip families as well as by tyrosine phosphorylation. In transformed cells, PCNA dissociates from the quaternary complex (14), implicating PCNA as a "sensor" for cell cycle progression at the early G1 to G1 restriction point transition. This hypothesis is supported by experiments in which PCNA and p21 were overexpressed in cells (16). Expression of the CDK2-interacting region (amino terminus) of p21 blocks cell cycle progression in a manner similar to overexpression of full-length p21. This effect cannot be antagonized by overexpression of PCNA. In contrast, overexpression of the PCNA-binding (carboxyl) terminus of p21 only weakly inhibits cell cycle progression, and this can be overcome by overexpression of PCNA. These data suggest that the negative regulatory effects of p21 can be mediated directly through CDKs or indirectly through PCNA. The interaction of cyclin D and PCNA is important for cell cycle (17, 18). Both the amino terminus (residues 2–64) and the carboxyl terminus (residues 197–228) of PCNA interact directly with D-type cyclins (19). Of particular note, the carboxyl terminal region of PCNA that interacts with cyclin D is almost identical with the region of PCNA that binds to DQ65–79 and was identified with the yeast two-hybrid screen.
We propose that the DQ65–79 peptide interacts with PCNA in the CDK/cyclin/p21/PCNA quaternary complex, sending negative signals that affect the early G1 to G1 restriction point transition. This proposed model is based largely on the following observations: PCNA is a component of the CDK/cyclin/p21/PCNA quaternary complex (13, 14, 15); DQ65–69 interacts with PCNA in activated T cell nuclei, and its anti-proliferative effects strongly correlate with the strength of this interaction; DQ65–79 blocks the early G1 to G1 restriction point transition but does not block DNA replication. With cellular transformation, the subunits rearrange in the CDK/cyclin/p21/PCNA quaternary complex (14) with an accompanying loss of cell cycle regulation. Our result demonstrates that the DQ65–79 peptide blocks early G1 to G1 restriction point transition as Rb is hypophosphorylated in activated T cells. Collectively, these findings support our model that the DQ65–79 peptide interacts with PCNA and negatively affects the "sensor" function of PCNA operative at the early G1 to G1 restriction point transition.
Rapamycin is a potent anti-proliferative macrolide that has
recently been approved for use in clinical transplantation
(20). Both rapamycin and DQ65–79 block cell cycle
progression at the early G1 to G1 restriction point
interface and act downstream of the IL-2 receptor (6). Rb
phosphorylation is blocked in T cells treated with rapamycin in a
similar fashion as DQ65–79 (Fig. 4
). Interestingly, rapamycin
down-regulates PCNA at the transcriptional level (21).
Most likely, both rapamycin and DQ65–79 negatively affect the
"sensor" function of PCNA at early G1 to G1
restriction point transition: rapamycin decreases the amount of PCNA
while DQ65–79 exerts its negative influence through protein-protein
interactions.
Peptides derived from HLA molecules modulate immune response both in vitro and in vivo, suggesting that conserved regions of HLA may have important cellular functions. A synthetic peptide corresponding to residues 75–84 of HLA-B2702 induces T cell anergy, perhaps via interaction with members of the heat shock protein 70 family (4). The DQ65–79 peptide, derived from an HLA class II sequence, is anti-proliferative, blocking cell cycle progression at the early G1 to G1 restriction point transition (6). In the present study, we show that the DQ65–79 peptide interacts with PCNA in the nucleus, providing an unexpected molecular explanation for this anti-proliferative effect. The DQ/PCNA interaction may send a negative signal through the CDK/cyclin/p21/PCNA quaternary complex to block cell cycle progression at the early G1 to G1 restriction point transition, adding new evidence to previous proposals that PCNA functions as a cell cycle "sensor" (13, 14, 15). These findings further suggest that conserved regions of HLA molecules mediate heretofore unappreciated cellular functions. Our observation identifies PCNA as a potential target for the design of novel synthetic compounds that may prove clinically useful as anti-proliferative agents by blocking cell cycle progression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alan M. Krensky, Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Room H306, Stanford, CA 94305-5208.
3 Abbreviations used in this paper: CDK, cyclin-dependent kinase; pol , polymerase ; PCNA, proliferating cell nuclear Ag; Rb, retinoblastoma.
Received for publication December 13, 1999. Accepted for publication March 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 domain of HLA-A2. Nature 325:625.[Medline]
holoenzyme. J. Biol. Chem. 272:2373.This article has been cited by other articles:
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  C. Dong, S.-C. Lyu, A. M. Krensky, and C. Clayberger DQ 65-79, A Peptide Derived from HLA Class II, Mimics p21 to Block T Cell Proliferation J. Immunol., November 15, 2003; 171(10): 5064 - 5070. [Abstract] [Full Text] [PDF]
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 L. Vallat, H. Magdelenat, H. Merle-Beral, P. Masdehors, G. Potocki de Montalk, F. Davi, M. Kruhoffer, L. Sabatier, T. F. Orntoft, and J. Delic The resistance of B-CLL cells to DNA damage-induced apoptosis defined by DNA microarrays Blood, June 1, 2003; 101(11): 4598 - 4606. [Abstract] [Full Text] [PDF]
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Y. Jiang, D. Chen, S.-C. Lyu, X. Ling, A. M. Krensky, and C. Clayberger DQ 65-79, a Peptide Derived from HLA Class II, Induces I{kappa}B Expression J. Immunol., April 1, 2002; 168(7): 3323 - 3328. [Abstract] [Full Text] [PDF]
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) or
HIS-DQ65–79 substituted with serine at residue 75 (HIS-75S; •).
B, Concentration-dependent binding of HIS-peptides to
PCNA in vitro. Binding of HIS-DQ65–79 (
