Interaction of PRP4 with Krüppel-Like
Factor 13 Regulates CCL5 Transcription
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J Immunol 2007; 178:7081-7087; ;
doi: 10.4049/jimmunol.178.11.7081
http://www.jimmunol.org/content/178/11/7081
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Copyright © 2007 by The American Association of
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References
Boli Huang, Yong-Tae Ahn, Lisa McPherson, Carol
Clayberger and Alan M. Krensky
The Journal of Immunology
Interaction of PRP4 with Krüppel-Like Factor 13 Regulates
CCL5 Transcription1
Boli Huang, Yong-Tae Ahn, Lisa McPherson, Carol Clayberger, and Alan M. Krensky2
A
fundamental question in inflammatory disease is how
immune cells move from the bloodstream to sites of disease. This process is central to the development of a
variety of acute and chronic immune-mediated diseases. Critical
components in this process are chemokines that direct the movement and infiltration of specific subsets of inflammatory cells to
the site of inflammation (1). This family of small proteins also
plays an important role in the control of leukocyte recruitment,
activation, and effector function, as well as hemopoiesis, the modulation of angiogenesis, and aspects of adaptive immunity (2– 6).
CCL5, a member of the C-C chemokine family, is a potent chemoattractant of T lymphocytes, monocytes, eosinophils, basophils,
and NK cells (7–11). CCL5 also activates T lymphocytes, causes
degranulation of basophils, and mediates a respiratory burst in eosinophils (12–14). Collectively, these functions implicate CCL5 as
an important mediator of both acute and chronic inflammation. The
chemokine receptor CCR5, which binds CCL5 and the related chemokines MIP-1␣ and MIP-1␤, serves as a coreceptor for HIV to
enter target cells (15–19). Thus, CCL5 has become an important
therapeutic target for immune-mediated diseases. The development of anti-inflammatory agents capable of blocking CCL5 expression may inhibit the generation of cellular infiltrate in autoimmunity and transplant rejection. In contrast, inducing CCL5
expression may be therapeutic for cancer and AIDS.
CCL5 is ubiquitously expressed in a variety of tissues under
different conditions (20 –23). In fibroblasts, epithelial cells, and
monocytes/macrophages, the expression of CCL5 is elevated
within hours of stimulation and is regulated by NF-␬B (24, 25). In
Department of Pediatrics, Stanford University School of Medicine, Stanford,
CA 94305
Received for publication December 22, 2006. Accepted for publication April 2, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a grant (to A.M.K.) from the National Institutes of
Health (NIH R37 DK35008-23). A.M.K. is the Shelagh Galligan Professor of
Pediatrics.
2
Address correspondence and reprint requests to Dr. Alan M. Krensky, Division of
Immunology and Transplantation Biology, Stanford University, 300 Pasteur Drive,
Stanford, CA 94305. E-mail address: [email protected]
www.jimmunol.org
contrast, in T lymphocytes, the induction of CCL5 occurs 3–5 days
after activation (26) and is regulated by a complex of proteins
recruited by Krüppel-like factor 13 (KLF13)3 (27, 28). These late
kinetics of expression help to amplify the immune response in both
time and space, and are consistent with the late expression of other
genes involved in T cell effector function including perforin,
granulysin, and granzymes A and B. KLF13 binds to the CTCCC
element of the human CCL5 promoter present in the A/B site (27).
Silencing of KLF13 expression in human T lymphocytes with
small interfering RNA (siRNA) decreases the expression of CCL5
mRNA and protein (28). Although KLF13 mRNA levels are similar in resting and activated T lymphocytes, KLF13 protein is only
expressed in actively proliferating and differentiating T lymphocytes (translational regulation), coincident with CCL5 gene expression (29). Interestingly, KLF13 is highly phosphorylated in activated T lymphocytes (27), suggesting that, like many transcription
factors, its activity is regulated by kinases. To identify binding
partners and potential regulators of KLF13 function, yeast twohybrid screening was performed in the present study. PRP4 kinase,
a member of the MAPK family, was found to bind KLF13 and to
regulate CCL5 expression in human T lymphocytes.
Materials and Methods
Antibodies
RFLAT-1 (C-19) Ab for supershifting KLF13 was purchased from Santa
Cruz Biotechnology. Alexa Fluor 488-conjugated anti-GFP and Alexa
Fluor 555 goat anti-mouse IgG2b(␥2b) were purchased from Molecular
Probes. Anti-hemagglutinin (HA) mouse mAb (clone 12CA5) was purchased from Roche Diagnostic Systems. Anti-V5 mouse mAb was purchased from Sigma-Aldrich. Anti-␣-actinin was obtained from Upstate
Biotechnology. Rabbit polyclonal antisera to KLF13 was produced as described previously (27). Rabbit polyclonal antisera to PRP4 was produced
by immunizing rabbits with synthetic peptides LKKLNDADPDDKFHC
(residues 736 –750) and CQRLPEDQRKKVHQLK (residues 962–977)
conjugated to keyhole limpet hemocyanin (Washington Biotechnology).
3
Abbreviations used in this paper: KLF13, Krüppel-like factor 13; HA, hemagglutinin; siRNA, small interfering RNA; RT-qPCR, real-time quantitative PCR; NLK,
Nemo-like kinase; GUS, ␤-glucuronidase; Brg-1, Brahma-related gene 1; Ct, threshold cycle; CBP, CREB binding protein; PCAF, p300/CBP-associated factor.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Activation of resting T lymphocytes initiates differentiation into mature effector cells over 3–7 days. The chemokine CCL5
(RANTES) and its major transcriptional regulator, Krüppel-like factor 13 (KLF13), are expressed late (3–5 days) after activation
in T lymphocytes. Using yeast two-hybrid screening of a human thymus cDNA library, PRP4, a serine/threonine protein kinase,
was identified as a KLF13-binding protein. Specific interaction of KLF13 and PRP4 was confirmed by reciprocal coimmunoprecipitation. PRP4 is expressed in PHA-stimulated human T lymphocytes from days 1 and 7 with a peak at day 3. Using an in vitro
kinase assay, it was found that PRP4 phosphorylates KLF13. Furthermore, although phosphorylation of KLF13 by PRP4 results
in lower binding affinity to the A/B site of the CCL5 promoter, coexpression of PRP4 and KLF13 increases nuclear localization
of KLF13 and CCL5 transcription. Finally, knock-down of PRP4 by small interfering RNA markedly decreases CCL5 expression
in T lymphocytes. Thus, PRP4-mediated phosphorylation of KLF13 plays a role in the regulation of CCL5 expression in T
lymphocytes. The Journal of Immunology, 2007, 178: 7081–7087.
7082
PRP4 REGULATES CCL5 TRANSCRIPTION
Plasmid constructs
pGL3-RP-luc was constructed by inserting ⫺195 to ⫹54 bp of the CCL5
promoter into the pGL3-basic vector (Promega). pSOS-KLF13 construct
was obtained by subcloning the PCR-amplified full-length KLF13 into
NcoI/SacI sites of the bait vector pSOS (Stratagene). pME-HA-PRP4,
which contains full-length PRP4, was a gift from Dr. M. Hagiwara (Tokyo
Medical and Dental University, Tokyo, Japan). pcDNA3.1-KLF13 and
pcDNA3.1/CT-GFP-KLF13 constructs were obtained by subcloning a PCR
product encoding full-length human KLF13 cDNA into the mammalian
expression vector pcDNA3.1⫺ and pcDNA3.1/CT-GFP (Invitrogen Life
Technologies), respectively. pcDNA3.1/V5-His-Nemo-like kinase (NLK)
was constructed by subcloning the full-length human NLK cDNA into
pcDNA3.1/V5-His vector (Invitrogen Life Technologies). pET28a-KLF13
and pET28a-KLF13 (1–263), used for expression of full-length or aa 264 –288
truncated recombinant KLF13, were obtained by subcloning the corresponding
PCR-amplified cDNA into pET28a⫹ vector (Invitrogen Life Technologies).
Yeast two-hybrid analysis
Cell culture, transfection, and luciferase assays
Human peripheral blood T lymphocytes were isolated from leukopacs
(Stanford Blood Bank) by negative selection (RosetteSep) according to the
manufacturer’s protocol (StemCell Technologies). T lymphocytes and
COS7 cells were cultured at 37°C with 5% CO2 in either RPMI 1640
medium (Irvine Scientific) or DMEM (Invitrogen Life Technologies) supplemented with 10% (v/v) FBS (HyClone), 2 mM L-glutamine, and 100
U/ml penicillin-streptomycin, respectively. T lymphocytes were stimulated
with 5 ␮g/ml PHA for up to 7 days. Transient transfection of COS7 cells was
performed using FuGENE 6 transfection reagent (Roche Diagnostics Systems)
according to the manufacturer’s recommendation. For luciferase reporter assays, COS7 cells (2.5 ⫻ 105) were transfected with pGL3-RP-luc (0.6 ␮g) plus
pcDNA3.1-KLF13 (0.3 ␮g) and/or pME-HA-PRP4 (0.3 ␮g). Corresponding
empty vectors were used to keep the total amount of DNA constant. A
pRL-TK plasmid (20 ng; Promega) encoding a Renilla luciferase gene was
included as an internal control. The cells were harvested and lysed 36-h posttransfection, and luciferase activity was determined using a Dual Luciferase
Assay Kit (Promega). Firefly luciferase activities were normalized to Renilla
activities to account for differences in transfection efficiency.
Immunoprecipitation and kinase assay
Transfected COS7 cells were lysed in buffer A containing 20 mM HEPES
(pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 1
mM DTT, and protease inhibitors. Lysates were clarified at 15,000 ⫻ g for
20 min at 4°C. Immunoprecipitations from cell lysates were conducted
using corresponding Ab along with protein A/G agarose beads. After incubating for 16 h at 4°C, immune complexes were collected by centrifugation and then washed two times with lysis buffer and two times with
PBS. For kinase assays, the immunoprecipitants were washed one more
time with kinase buffer (25 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM
EGTA, and 1 mM DTT). In the kinase reaction, His-tagged full-length or
aa 264 –288-truncated KLF13 was used as substrate. Purification of recombinant KLF13 protein through a Ni⫹ column was described previously
(27). The immunocomplexes were incubated at 30°C in kinase buffer supplemented with 1 mM ATP, 1 ␮Ci [␥-32P]ATP, and 1 ␮g of recombinant
KLF13 in a volume of 30 ␮l for 15 min. The reaction was terminated by
the addition of SDS-PAGE loading buffer and the samples were subjected
to SDS-PAGE. Phosphorylated KLF13 was visualized by autoradiography.
For EMSA, phosphorylated recombinant KLF13 was made using cold ATP
instead of [␥-32P]ATP in the kinase reaction.
Northern blot
Total RNA was extracted from human T lymphocytes using the RNeasy
Mini Kit according to the manufacturer’s instructions (Qiagen). Twenty
micrograms of total RNA was used for Northern blotting as previously
described (30). The membrane was hybridized with 32P-labeled PRP4
cDNA, stripped, and then reprobed with 32P-labeled 28S rRNA antisense
oligonucleotide to confirm equal loading and transfer. Relative quantification was performed using densitometry.
FIGURE 1. KLF13 interacts with PRP4. A, Lysates of COS7 cells transfected with pcDNA3.1-KLF13 (KLF13) along with either pME-HA (vector) or pME-HA-PRP4 (HA-PRP4) were immunoprecipitated with anti-HA
mouse mAb. B, Lysates of COS7 cells transfected with HA-PRP4, along
with either pcDNA3.1 (vector) or KLF13, were immunoprecipitated with
anti-KLF13 antisera. Bound proteins were analyzed by Western blot using
the indicated Abs. Equal input of KLF13 (A) and PRP4 (B) was confirmed
by Western blot of unprecipitated cell lysates.
Western blot
T lymphocytes were lysed in buffer A as described above. T cell lysates or
immunoprecipitants from transfected COS7 cells were subjected to SDSPAGE and the membrane was hybridized with Abs against KLF13 or
PRP4. ECL Western blotting detection reagents were used for detection
(Amersham Biosciences). Loading and transfer efficiency were confirmed
by blotting with a mouse Ab against ␣-actinin.
EMSA
A double-stranded oligonucleotide corresponding to the A/B site of the
CCL5 promoter was used as a probe for EMSA as previously described
(27). The labeled probe (20,000 cpm) was incubated with 1 ␮g of recombinant KLF13 from the kinase reaction, 1.5 ␮g of poly(dI:dC), 10 mM
Tris-HCl (pH 7.5), 80 mM NaCl, 1 mM EDTA, 1 mM DTT, and 5%
glycerol for 20 min at room temperature. EMSA was also performed using
immunoprecipitants from COS7 cells transfected with either empty vector
(vector) or pME-HA-PRP4 (PRP4) in the absence of KLF13 as additional
negative controls. For supershift assays, 2 ␮g of Ab was added to the
binding mixture. DNA-protein complexes were analyzed on a 5% nondenaturing polyacrylamide gel in 0.5 ⫻ Tris-borate-EDTA buffer.
Cellular localization experiments
To visualize the intracellular localization of KLF13 and PRP4, COS7 cells
(2 ⫻ 104) grown on tissue culture glass slides were transfected with
pcDNA3.1-GFP-KLF13 (0.2 ␮g) and pME-HA-PRP4 (0.2 ␮g), either
alone or in combination using FuGene 6 transfection reagent. Corresponding empty vector was used as a control. After 24 h, the transfected cells
were fixed and permeabilized. KLF13 and PRP4 were detected by immunofluorescence as described by Kojima et al. (31) using Alexa Fluor 488conjugated anti-GFP (1/2000 dilution) for GFP-tagged KLF13 and anti-HA
mouse mAb (1/2, 000 dilution) for HA-tagged PRP4. Secondary Ab conjugated to Alexa Fluor 555 goat anti-mouse IgG2b(␥2b) (1/1,000; Invitrogen Life Technologies) was used for double labeling of HA-tagged PRP4.
The nuclei were visualized using Hoechst 33342 (1/10,000; Invitrogen Life
Technologies) staining. The cellular localization of KLF13 and PRP4 proteins was monitored using an immunofluorescence microscope.
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The CytoTrap two-hybrid system (Stratagene) was used to identify proteins
that bind to KLF13 using pSOS-KLF13 as the bait construct. This construct
was cotransfected into a mutant yeast strain cdc25H␣ with a CytoTrap XR
Human Thymus cDNA Library cloned in pMyr vector following the manufacturer’s protocol. Plasmids were extracted from putative positive colonies and transformed into Escherichia coli to be further analyzed by DNA
sequencing. Only peptide sequences that were in frame in pMyr were considered for further validation.
The Journal of Immunology
7083
siRNA and real-time quantitative PCR (RT-qPCR)
Knock-down of PRP4 mRNA was achieved using double-stranded siRNA
oligonucleotides obtained from Qiagen. Nonsilencing siRNA (5⬘-UUCUC
CGAACGUGUCACGUdTdT-3⬘), which has no homology to any known
mammalian gene, was used as a negative control. A total of 1.5 ␮g of
siRNA was nucleofected into 5 ⫻ 106 freshly isolated T lymphocytes using
the Human T Cell Nucleofector Kit per the manufacturer’s instruction
(Amaxa). PHA was added 4 h after nucleofection to achieve a final concentration of 5 ␮g/ml. Cells were incubated for an additional 30 h and
harvested for both nuclear extract preparation and RNA isolation. To measure gene expression, cDNA was made from total RNA using Superscript
II with random hexamers (Invitrogen Life Technologies) for use in RTqPCR with the GeneAmp 7900 Sequence Detection System (Applied Biosystems). NLK primers were used in RT-qPCR to determine the specificity
of PRP4 siRNAs, and ␤-glucuronidase (GUS) was used for normalizing
RT-qPCR results. Primers for CCL5 and NLK were synthesized by Elim
Biopharmaceuticals, and primers for PRP4 and GUS were purchased from
Applied Biosystems. All RT-qPCR was performed in triplicate using either
SYBR green or TaqMan Universal PCR Master Mix (Applied Biosystems).
The expression level of a gene in a given sample was represented as
2⫺⌬⌬Ct, where ⌬⌬Ct ⫽ (⌬Ct(silencing)) ⫺ (⌬Ct(non-silencing)) and ⌬Ct ⫽
(Ct(sample) ⫺ Ct(Gus)). Values represent the fold change of target gene
expression from PRP4 siRNA-transfected vs nonsilencing siRNA-transfected primary T lymphocytes.
ELISA
CCL5 protein levels were measured in the culture supernatant using the
Endogen Human RANTES ELISA Kit (Pierce) according to the manufacturer’s instruction.
FIGURE 3. PRP4 phosphorylates KLF13 and disrupts the binding of
KLF13 to the CCL5 promoter A/B site. COS7 cells were transfected with
pcDNA3.1/V5-His-NLK (V5-NLK), pME-HA-PRP4 (HA-PRP4), or corresponding empty vectors, as indicated, and immunoprecipitated with antiHA or anti-V5 Ab. A, Recombinant KLF13 or KLF13 (1–263) were phosphorylated in a kinase reaction in the presence of [␥-32P]ATP with either
vector, V5-NLK, or HA-PRP4 immunoprecipitants, as indicated. and subjected to SDS-PAGE followed by autoradiography. B, Immunoprecipitants
from COS7 cells transfected with vector or HA-PRP4 were used to phosphorylate recombinant KLF13 in the presence of cold ATP, and the products were subjected to EMSA. Kinase reactions lacking KLF13 substrate
were also analyzed as controls. KLF13-specific Ab was used to supershift.
Results
PRP4 binds KLF13 in human thymocytes
The CytoTrap yeast two-hybrid system was used to screen a human thymus library for proteins that bind to KLF13. Approximately 2 ⫻ 106 transformants were screened with a pSOS-KLF13
construct, and 19 putative positive clones were identified. Comparison of cDNA sequences of the clones with the GenBank database revealed that one of them is the human homolog of the yeast
serine-threonine kinase PRP4. To further confirm the physical interaction between KLF13 and PRP4, reciprocal coimmunoprecipitation experiments were performed. COS7 cells were cotransfected
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FIGURE 2. Expression of PRP4 in resting and PHA-activated T lymphocytes. A, Northern blot of PRP4 mRNA. Blots were sequentially hybridized with 32P-labeled human PRP4 cDNA and 28S rRNA oligonucleotide probe. Bar graph, Quantification of the relative abundance of PRP4
mRNA normalized to 28S rRNA. B, Western blot of PRP4 protein. PRP4specific antisera were used to detect protein, whereas ␣-actinin Ab was
used as an internal control. Bar graph, Quantification of the amount of
PRP4 protein normalized to ␣-actinin. Results are representative of four
similar experiments.
7084
PRP4 REGULATES CCL5 TRANSCRIPTION
levels of PRP4 mRNA were detected by Northern blot in resting T
lymphocytes (Fig. 2A). mRNA levels increased 5-fold 1 day after
the activation of T lymphocytes with PHA and, by day 3, had
increased ⬃20-fold. PRP4 mRNA decreased on day 5 and, by day
7, PRP4 mRNA dropped to the same level seen on day 1. PRP4specific rabbit antisera were generated to monitor PRP4 protein
expression during T lymphocyte activation by Western blot (Fig.
2B). Parallel to the mRNA results, only trace amounts of PRP4
protein were present in resting T lymphocytes. PRP4 protein increased 1 day after activation and continued to increase with a
peak of 9-fold induction observed by day 3. Protein levels decreased on days 5 and 7, consistent with mRNA expression. Thus,
PRP4 kinase is expressed in human T lymphocytes and displays an
activation kinetic peaking in expression at day 3.
with pME-HA-PRP4 construct and pcDNA3.1-KLF13. Replacement of one expression plasmid with its corresponding empty vector was used to show specificity of coimmunoprecipitation. Only
HA-PRP4-transfected COS7 lysates immunoprecipitated with an
anti-HA Ab against tagged PRP4 pulled down KLF13 (Fig. 1A),
while the reciprocal experiment using a KLF13 Ab pulled down
PRP4 (Fig. 1B). These data confirm that KLF13 binds PRP4.
PRP4, a MAPK, is expressed in T lymphocytes
T lymphocytes were isolated from peripheral blood and activated
with the mitogen PHA. PRP4 mRNA and protein were measured
in resting T lymphocytes and through 7 days after activation. Low
FIGURE 5. PRP4 increases the nuclear localization of KLF13. COS7
cells were transfected with either pcDN
A3.1/CT-GFP-KLF13 (GFP-KLF13),
or pME-HA-PRP4 (HA-PRP4), or in
combination (GFP-KLF13 ⫹ HAPRP4). Cells transfected with corresponding amounts of pcDNA3.1 and
pME-HA were used as negative control
(Vector). After 24 h, GFP-KLF13 and
HA-PRP4 were stained with anti-GFP
Ab and anti-HA Ab, respectively, and
detected using immunofluorescence
microscopy. Nuclei were visualized by
Hoechst staining. Composite shows the
overlapped image of anti-GFP Ab, antiHA Ab, and Hoechst stained images.
Arrow, Cell expressing only GFPKLF13 that shows a different pattern of
cellular distribution compared with
other cells in this field that coexpress
both GFP-KLF13 and HA-PRP4.
PRP4 phosphorylates KLF13, decreasing its binding affinity to
the CCL5 promoter A/B site
KLF13 is the major transcription factor regulating CCL5 expression in T lymphocytes (27, 28). KLF13 is expressed late (days
3–5) after T lymphocyte activation, is rapidly phosphorylated, and
is present in both the nucleus and the cytosol (27, 32). Because the
protein expression patterns of KLF13 and PRP4 are similar, we
tested whether PRP4 and NLK, another MAPK family member,
could phosphorylate KLF13 (Fig. 3A). The immunoprecipitants
derived from PRP4- or NLK-overexpressing COS7 cells were used
to phosphorylate recombinant KLF13 in a kinase assay, with immunoprecipitants from corresponding vector-transfected cells used
as a negative control. Under these conditions, immunoprecipitated
PRP4 phosphorylated both itself and KLF13 (Fig. 3A, lane 4). In
contrast, immunoprecipitated NLK phosphorylated itself, but not
KLF13 (Fig. 3A, lane 2). We previously found that NLK was
recruited to the CCL5 promoter by KLF13 and subsequently phosphorylates serine 10 of histone H3 upon T lymphocyte activation
(28). These results suggest that PRP4 and NLK regulate CCL5
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FIGURE 4. PRP4 and KLF13 synergize to regulate CCL5 transcription.
pGL3-RP-luc and pRL-TK were cotransfected with pcDNA3.1-KLF13
(KLF13) and/or pME-HA-PRP4 (PRP4) into COS7 cells. Results are
shown as relative luciferase activity with the basal luciferase activity (vector) set to 1. The data are expressed as the average ⫾ SD from three
independent experiments. The p values compared with vector control are
indicated.
The Journal of Immunology
7085
KLF13 and PRP4 synergistically regulate CCL5 expression
Reporter gene assays were performed to examine the effect of
PRP4 on CCL5 transcription (Fig. 4). The CCL5 promoter luciferase reporter construct pGL3-RP was transfected into COS7 cells
along with KLF13 and/or PRP4 expression constructs. Compared
with vector control, expression of KLF13 caused a 2.8-fold induction ( p ⬍ 0.0001) in CCL5 reporter gene activity, while expression of PRP4 caused a 1.4-fold increase ( p ⫽ 0.01). However,
coexpression of KLF13 and PRP4 resulted in a 5.2-fold increase
( p ⬍ 0.0001) in CCL5 promoter activity. Of note, we detected
endogenous PRP4 in COS7 cells (data not shown), explaining the
modest increase in CCL5 promoter activity in the presence of
transfected PRP4. These results indicate that PRP4 synergizes with
KLF13 to regulate CCL5 expression in vitro.
PRP4 increases the nuclear localization of KLF13
To further investigate the role of PRP4 in regulating CCL5 expression, the effect of PRP4 on intracellular localization of KLF13
was assessed. COS7 cells were transiently transfected with expression vectors corresponding to empty vector, GFP-KLF13, HAPRP4, or a combination of GFP-KLF13 and HA-PRP4. No signal
was detected when cells were transfected with pcDNA3.1, and
only a small amount of background signal was observed throughout the cell following transfection of the pME-HA vector (Fig. 5).
In the absence of PRP4, overexpressed KLF13 protein was detected in the nuclear compartment of all KLF13-transfected COS7
cells, while 45–50% of these cells also had KLF13 in the cytoplasm (Fig. 5, GFP-KLF13/␣-GFPAb). In contrast, when KLF13
was coexpressed with PRP4, KLF13 resided almost exclusively in
the nucleus, indicating that PRP4 regulates the nuclear translocation of KLF13 (Fig. 5, GFP-KLF13 ⫹ HA-PRP4/␣-GFPAb). The
arrows in the frames transfected with GFP-KLF13 ⫹ HA-PRP4
(Fig. 5) mark a single cell that expresses KLF13, but not PRP4. It
is evident that, in the absence of PRP4, overexpressed KLF13 is
distributed both in the nuclei and cytoplasm of the cell. In contrast,
overexpressed KLF13 resides exclusively in the nuclei of the other
cells in the same field that is coexpressing both KLF13 and PRP4,
supporting the role of PRP4 in KLF13 nuclear translocation. Over-
FIGURE 6. Knock-down of PRP4 reduces CCL5 expression in T lymphocytes. A, Levels of CCL5, PRP4, and NLK mRNA after nucleofection
of nonsilencing and PRP4 (1 and 2) siRNAs into resting human T lymphocytes followed by 30 h of PHA activation. Average ⫾ SD of three
experiments are shown. B, Protein levels of PRP4 and CCL5 in human T
lymphocytes after treatment with nonsilencing or PRP4 siRNAs. Left,
Western blot analysis of PRP4 protein. PRP4-specific antisera were used to
determine the amounts of protein, whereas ␣-actinin Ab was used to normalize for protein loading. The graph shows the ratio of PRP4:␣-actinin
expression. Right: CCL5 in culture supernatants measured by ELISA. Data
represent the mean ⫾ SD of three triplicates.
expressed PRP4 is predominantly expressed in the nucleus of the
cell, regardless of the presence or absence of overexpressed
KLF13 (Fig. 5, HA-PRP4/␣HAAb). Frames of Fig. 5 in each row
shows nuclei visualized by Hoechst staining. Composite was generated by merging images from frames 1–3 (Fig. 5) in each row to
better visualize colocalization.
Knock-down of PRP4 decreases CCL5 expression in human T
lymphocytes
To confirm the role of PRP4 in regulating CCL5 expression in T
lymphocytes, two sets of PRP4-specific siRNAs were nucleofected
into resting human T lymphocytes. The cells were then stimulated
with PHA, and the expression of CCL5 was measured after 30 h
using RT-qPCR and ELISA. PRP4 mRNA (Fig. 6A) and protein
(Fig. 6B) were suppressed ⬎65% by both sets of siRNA. In contrast, PRP4-specific siRNAs caused only 10 –15% suppression of
NLK mRNA expression (Fig. 6A), demonstrating the specificity of
the siRNAs. Moreover, both sets of PRP4 siRNA repressed CCL5
expression, with ⬃55– 60% reduction in mRNA transcription and
30 –35% reduction in protein expression (Fig. 6). Thus, PRP4 regulates CCL5 expression in T lymphocytes.
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expression by phosphorylating different target proteins. Immunoprecipitated PRP4 also phosphorylated KLF13 (1–263) that has 25
aa deleted from the C terminus (Fig. 3A, lane 6). However, phosphorylation was less robust, suggesting that KLF13 may contain
multiple PRP4 phosphorylation sites, with one or more sites in the
truncated region.
To test whether the phosphorylation of KLF13 by PRP4 affects
its interaction with the CCL5 promoter, EMSA was performed
using a ␥-32P-end-labeled oligonucleotide derived from the A/B
region of the CCL5 promoter. Full-length recombinant KLF13 was
phosphorylated by immunoprecipitated PRP4 obtained from
transfected COS7 cell lysate using unlabeled ATP, with immunoprecipitant from vector-transfected COS7 cell lysate serving
as control. An upper doublet and three lower bands were observed. Although the upper doublet most likely corresponds to
the full-length KLF13 bound to probe, the lower bands may
result from the degradation of the KLF13 substrate during the
kinase reaction. All of these DNA-protein complexes were supershifted upon the addition of anti-KLF13 Ab (Fig. 3B), but
not with anti-p50 Ab (data not shown), indicating the specificity
of binding. In the presence of PRP4, there is a ⬃50% decrease
in KLF13 bound to the CCL5 promoter A/B site probe (Fig.
3B). Thus, phosphorylation of KLF13 by PRP4 results in lower
binding affinity to the CCL5 promoter compared with nonphosphorylated KLF13.
7086
Discussion
is unable to phosphorylate KLF13, did not enhance transcription
(data not shown). This indicates that the N-terminal region of
PRP4 is not only important for the phosphorylation of KLF13, but
also plays a role in regulating CCL5 expression. The apparent
dichotomy between decreased KLF13 DNA binding induced by
PRP4-mediated phosphorylation and increased CCL5 expression
remains unclear, but may be related to the complexity of the CCL5
enhancesome (28). In this regard, Song et al. (40) reported that the
transcriptional coactivators p300 CREB-binding protein (CBP)
and p300-CBP-associated factor (PCAF) act cooperatively in stimulating KLF13 transcriptional activity. Nevertheless, p300/CBP
and PCAF acetylate specific lysine residues in the zinc finger
DNA-binding domain of KLF13 which disrupt KLF13 DNA binding. In comparison, we found that the mutation of two amino acids
in the same zinc finger DNA-binding domain of KLF13 causes a
dramatic increase in CCL5 reporter gene activity, although the
mutated KLF13 bound to the CCL5 A/B site with much lower
affinity (data not shown). Moreover, we recently confirmed that
KLF13 recruits p300/CBP and PCAF to the CCL5 promoter in
activated T lymphocytes (28). Studies are currently underway to
determine whether PRP4 mediates changes in the acetylation states
of KLF13 in concert with other acetyltransferase proteins, such as
p300/CBP and PCAF and, therefore, reduces its DNA-binding affinity. We hypothesize that the phosphorylation of KLF13 by
PRP4 may facilitate the assembly or disassembly of the multiprotein complex at the CCL5 promoter, enabling PRP4 to further
modify transcriptional coactivators or repressors by phosphorylation, as many ERK/MAPK and cyclic-dependent kinases have
been shown to associate with and phosphorylate transcription factors or transcriptional coactivators (41).
Control of CCL5 expression in T lymphocytes is complex.
CCL5 transcription is controlled by an enhancesome composed of
different factors at various times after T lymphocyte activation
(28). KLF13, a sequence-specific DNA transcription factor, coordinates the induction of CCL5 expression in T lymphocytes by
ordered recruitment of proteins to the CCL5 promoter, including
Brahma-related gene 1 (Brg-1) (28). Brg-1 is an ATPase subunit of
the SWI-SNF chromatin-remodeling complexes (42). It is recruited to chromatin by direct interactions with DNA-binding proteins (43). Interestingly, Dellaire et al. (37) demonstrated that
Brg-1 interacts with the hypophosphorylated form of PRP4 in a
transcription-dependent manner and appears to be a PRP4 substrate in vitro. Nuclear receptor corepressor, a component of the
nuclear hormone corepressor complex, also interacts in vivo with
human PRP4. In summary, PRP4 binds to and phosphorylates
KLF13, enhancing CCL5 expression. Therefore, PRP4 is an important component of the enhancesome assembling over time at
the CCL5 promoter in activated T lymphocytes.
Disclosures
The authors have no financial conflict of interest.
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PRP4 REGULATES CCL5 TRANSCRIPTION
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