Factor 13 Regulates CCL5 Transcription Interaction of PRP4

Interaction of PRP4 with Krüppel-Like Factor 13 Regulates CCL5 Transcription Boli Huang, Yong-Tae Ahn, Lisa McPherson, Carol Clayberger and Alan M. Krensky This information is current as of September 28, 2021. J Immunol 2007; 178:7081-7087; ; doi: 10.4049/jimmunol.178.11.7081 http://www.jimmunol.org/content/178/11/7081 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Interaction of PRP4 with Kru¨ppel-Like Factor 13 Regulates CCL5 Transcription1

Boli Huang, Yong-Tae Ahn, Lisa McPherson, Carol Clayberger, and Alan M. Krensky2

Activation of resting T lymphocytes initiates differentiation into mature effector cells over 3–7 days. The chemokine CCL5 (RANTES) and its major transcriptional regulator, Kru¨ppel-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 Downloaded from 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.

fundamental question in inflammatory disease is how contrast, in T lymphocytes, the induction of CCL5 occurs 3–5 days immune cells move from the bloodstream to sites of dis- after activation (26) and is regulated by a complex of proteins ease. This process is central to the development of a recruited by Kru¨ppel-like factor 13 (KLF13)3 (27, 28). These late A http://www.jimmunol.org/ variety of acute and chronic immune-mediated diseases. Critical kinetics of expression help to amplify the immune response in both components in this process are chemokines that direct the move- time and space, and are consistent with the late expression of other ment and infiltration of specific subsets of inflammatory cells to genes involved in T cell effector function including perforin, the site of inflammation (1). This family of small proteins also granulysin, and granzymes A and B. KLF13 binds to the CTCCC plays an important role in the control of leukocyte recruitment, element of the human CCL5 promoter present in the A/B site (27). activation, and effector function, as well as hemopoiesis, the mod- Silencing of KLF13 expression in human T lymphocytes with ulation of angiogenesis, and aspects of adaptive immunity (2–6). small interfering RNA (siRNA) decreases the expression of CCL5 CCL5, a member of the C-C chemokine family, is a potent che- mRNA and protein (28). Although KLF13 mRNA levels are sim- moattractant of T lymphocytes, monocytes, eosinophils, basophils, ilar in resting and activated T lymphocytes, KLF13 protein is only by guest on September 28, 2021 and NK cells (7–11). CCL5 also activates T lymphocytes, causes expressed in actively proliferating and differentiating T lympho- degranulation of basophils, and mediates a respiratory burst in eo- cytes (translational regulation), coincident with CCL5 gene expres- sinophils (12–14). Collectively, these functions implicate CCL5 as sion (29). Interestingly, KLF13 is highly phosphorylated in acti- an important mediator of both acute and chronic inflammation. The vated T lymphocytes (27), suggesting that, like many transcription chemokine receptor CCR5, which binds CCL5 and the related che- factors, its activity is regulated by kinases. To identify binding mokines MIP-1␣ and MIP-1␤, serves as a coreceptor for HIV to partners and potential regulators of KLF13 function, yeast two- enter target cells (15–19). Thus, CCL5 has become an important hybrid screening was performed in the present study. PRP4 kinase, therapeutic target for immune-mediated diseases. The develop- a member of the MAPK family, was found to bind KLF13 and to ment of anti-inflammatory agents capable of blocking CCL5 ex- regulate CCL5 expression in human T lymphocytes. pression may inhibit the generation of cellular infiltrate in auto- immunity and transplant rejection. In contrast, inducing CCL5 Materials and Methods expression may be therapeutic for cancer and AIDS. Antibodies CCL5 is ubiquitously expressed in a variety of tissues under different conditions (20–23). In fibroblasts, epithelial cells, and RFLAT-1 (C-19) Ab for supershifting KLF13 was purchased from Santa Cruz Biotechnology. Alexa Fluor 488-conjugated anti-GFP and Alexa monocytes/macrophages, the expression of CCL5 is elevated Fluor 555 goat anti-mouse IgG2b(␥2b) were purchased from Molecular within hours of stimulation and is regulated by NF-␬B (24, 25). In 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 Department of Pediatrics, Stanford University School of Medicine, Stanford, Biotechnology. Rabbit polyclonal antisera to KLF13 was produced as de- CA 94305 scribed previously (27). Rabbit polyclonal antisera to PRP4 was produced by immunizing rabbits with synthetic peptides LKKLNDADPDDKFHC Received for publication December 22, 2006. Accepted for publication April 2, 2007. (residues 736–750) and CQRLPEDQRKKVHQLK (residues 962–977) The costs of publication of this article were defrayed in part by the payment of page conjugated to keyhole limpet hemocyanin (Washington Biotechnology). 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 3 Abbreviations used in this paper: KLF13, Kru¨ppel-like factor 13; HA, hemagglu- Health (NIH R37 DK35008-23). A.M.K. is the Shelagh Galligan Professor of tinin; siRNA, small interfering RNA; RT-qPCR, real-time quantitative PCR; NLK, Pediatrics. Nemo-like kinase; GUS, ␤-glucuronidase; Brg-1, Brahma-related gene 1; Ct, thresh- 2 Address correspondence and reprint requests to Dr. Alan M. Krensky, Division of old cycle; CBP, CREB binding protein; PCAF, p300/CBP-associated factor. Immunology and Transplantation Biology, Stanford University, 300 Pasteur Drive, Stanford, CA 94305. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org 7082 PRP4 REGULATES CCL5 TRANSCRIPTION

Plasmid constructs pGL3-RP-luc was constructed by inserting Ϫ195 to ϩ54 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 pcDNA3.1Ϫ 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 pET28aϩ vector (Invitrogen Life Technologies). Yeast two-hybrid analysis 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 man- Downloaded from ufacturer’s protocol. Plasmids were extracted from putative positive colo- nies and transformed into Escherichia coli to be further analyzed by DNA sequencing. Only peptide sequences that were in frame in pMyr were con- sidered for further validation. Cell culture, transfection, and luciferase assays FIGURE 1. KLF13 interacts with PRP4. A, Lysates of COS7 cells transfected with pcDNA3.1-KLF13 (KLF13) along with either pME-HA (vec- Human peripheral blood T lymphocytes were isolated from leukopacs tor) or pME-HA-PRP4 (HA-PRP4) were immunoprecipitated with anti-HA http://www.jimmunol.org/ (Stanford Blood Bank) by negative selection (RosetteSep) according to the mouse mAb. B, Lysates of COS7 cells transfected with HA-PRP4, along manufacturer’s protocol (StemCell Technologies). T lymphocytes and with either pcDNA3.1 (vector) or KLF13, were immunoprecipitated with COS7 cells were cultured at 37°C with 5% CO2 in either RPMI 1640 medium (Irvine Scientific) or DMEM (Invitrogen Life Technologies) sup- anti-KLF13 antisera. Bound proteins were analyzed by Western blot using plemented with 10% (v/v) FBS (HyClone), 2 mM L-glutamine, and 100 the indicated Abs. Equal input of KLF13 (A) and PRP4 (B) was confirmed U/ml penicillin-streptomycin, respectively. T lymphocytes were stimulated by Western blot of unprecipitated cell lysates. 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 ϫ 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 Western blot by guest on September 28, 2021 empty vectors were used to keep the total amount of DNA constant. A T lymphocytes were lysed in buffer A as described above. T cell lysates or pRL-TK plasmid (20 ng; Promega) encoding a Renilla luciferase gene was immunoprecipitants from transfected COS7 cells were subjected to SDS- included as an internal control. The cells were harvested and lysed 36-h post- PAGE and the membrane was hybridized with Abs against KLF13 or transfection, and luciferase activity was determined using a Dual Luciferase PRP4. ECL Western blotting detection reagents were used for detection Assay Kit (Promega). Firefly luciferase activities were normalized to Renilla (Amersham Biosciences). Loading and transfer efficiency were confirmed activities to account for differences in transfection efficiency. by blotting with a mouse Ab against ␣-actinin. Immunoprecipitation and kinase assay EMSA Transfected COS7 cells were lysed in buffer A containing 20 mM HEPES A double-stranded oligonucleotide corresponding to the A/B site of the (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 ϫ g for CCL5 promoter was used as a probe for EMSA as previously described 20 min at 4°C. Immunoprecipitations from cell lysates were conducted (27). The labeled probe (20,000 cpm) was incubated with 1 ␮g of recom- using corresponding Ab along with protein A/G agarose beads. After in- binant KLF13 from the kinase reaction, 1.5 ␮g of poly(dI:dC), 10 mM cubating for 16 h at 4°C, immune complexes were collected by centrifu- Tris-HCl (pH 7.5), 80 mM NaCl, 1 mM EDTA, 1 mM DTT, and 5% gation and then washed two times with lysis buffer and two times with glycerol for 20 min at room temperature. EMSA was also performed using PBS. For kinase assays, the immunoprecipitants were washed one more immunoprecipitants from COS7 cells transfected with either empty vector (vector) or pME-HA-PRP4 (PRP4) in the absence of KLF13 as additional time with kinase buffer (25 mM Tris-HCl (pH 7.6), 10 mM MgCl2,1mM EGTA, and 1 mM DTT). In the kinase reaction, His-tagged full-length or negative controls. For supershift assays, 2 ␮g of Ab was added to the aa 264–288-truncated KLF13 was used as substrate. Purification of recom- binding mixture. DNA-protein complexes were analyzed on a 5% nonde- binant KLF13 protein through a Niϩ column was described previously naturing polyacrylamide gel in 0.5 ϫ Tris-borate-EDTA buffer. (27). The immunocomplexes were incubated at 30°C in kinase buffer sup- plemented with 1 mM ATP, 1 ␮Ci [␥-32P]ATP, and 1 ␮g of recombinant Cellular localization experiments ␮ KLF13 in a volume of 30 l for 15 min. The reaction was terminated by To visualize the intracellular localization of KLF13 and PRP4, COS7 cells the addition of SDS-PAGE loading buffer and the samples were subjected (2 ϫ 104) grown on tissue culture glass slides were transfected with to SDS-PAGE. Phosphorylated KLF13 was visualized by autoradiography. pcDNA3.1-GFP-KLF13 (0.2 ␮g) and pME-HA-PRP4 (0.2 ␮g), either For EMSA, phosphorylated recombinant KLF13 was made using cold ATP alone or in combination using FuGene 6 transfection reagent. Correspond- ␥ 32 instead of [ - P]ATP in the kinase reaction. ing empty vector was used as a control. After 24 h, the transfected cells Northern blot were fixed and permeabilized. KLF13 and PRP4 were detected by immunofluorescence as described by Kojima et al. (31) using Alexa Fluor 488- Total RNA was extracted from human T lymphocytes using the RNeasy conjugated anti-GFP (1/2000 dilution) for GFP-tagged KLF13 and anti-HA Mini Kit according to the manufacturer’s instructions (Qiagen). Twenty mouse mAb (1/2, 000 dilution) for HA-tagged PRP4. Secondary Ab con- micrograms of total RNA was used for Northern blotting as previously jugated to Alexa Fluor 555 goat anti-mouse IgG2b(␥2b) (1/1,000; Invitro- described (30). The membrane was hybridized with 32P-labeled PRP4 gen Life Technologies) was used for double labeling of HA-tagged PRP4. cDNA, stripped, and then reprobed with 32P-labeled 28S rRNA antisense The nuclei were visualized using Hoechst 33342 (1/10,000; Invitrogen Life oligonucleotide to confirm equal loading and transfer. Relative quantifica- Technologies) staining. The cellular localization of KLF13 and PRP4 pro- tion was performed using densitometry. teins was monitored using an immunofluorescence microscope. The Journal of Immunology 7083 Downloaded from http://www.jimmunol.org/

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. PRP4-

speciﬁc antisera were used to detect protein, whereas ␣-actinin Ab was by guest on September 28, 2021 used as an internal control. Bar graph, Quantiﬁcation of the amount of PRP4 protein normalized to ␣-actinin. Results are representative of four similar experiments.

siRNA and real-time quantitative PCR (RT-qPCR) Knock-down of PRP4 mRNA was achieved using double-stranded siRNA FIGURE 3. PRP4 phosphorylates KLF13 and disrupts the binding of oligonucleotides obtained from Qiagen. Nonsilencing siRNA (5Ј-UUCUC KLF13 to the CCL5 promoter A/B site. COS7 cells were transfected with CGAACGUGUCACGUdTdT-3Ј), which has no homology to any known pcDNA3.1/V5-His-NLK (V5-NLK), pME-HA-PRP4 (HA-PRP4), or cor- mammalian gene, was used as a negative control. A total of 1.5 ␮gof responding empty vectors, as indicated, and immunoprecipitated with anti- 6 siRNA was nucleofected into 5 ϫ 10 freshly isolated T lymphocytes using HA or anti-V5 Ab. A, Recombinant KLF13 or KLF13 (1–263) were phos- the Human T Cell Nucleofector Kit per the manufacturer’s instruction phorylated in a kinase reaction in the presence of [␥-32P]ATP with either (Amaxa). PHA was added 4 h after nucleofection to achieve a final con- vector, V5-NLK, or HA-PRP4 immunoprecipitants, as indicated. and sub- centration of 5 ␮g/ml. Cells were incubated for an additional 30 h and jected to SDS-PAGE followed by autoradiography. B, Immunoprecipitants harvested for both nuclear extract preparation and RNA isolation. To mea- sure gene expression, cDNA was made from total RNA using Superscript from COS7 cells transfected with vector or HA-PRP4 were used to phos- II with random hexamers (Invitrogen Life Technologies) for use in RT- phorylate recombinant KLF13 in the presence of cold ATP, and the prod- qPCR with the GeneAmp 7900 Sequence Detection System (Applied Bio- ucts were subjected to EMSA. Kinase reactions lacking KLF13 substrate systems). NLK primers were used in RT-qPCR to determine the specificity were also analyzed as controls. KLF13-specific Ab was used to supershift. 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 Results Applied Biosystems. All RT-qPCR was performed in triplicate using either PRP4 binds KLF13 in human thymocytes SYBR green or TaqMan Universal PCR Master Mix (Applied Biosystems). The expression level of a gene in a given sample was represented as The CytoTrap yeast two-hybrid system was used to screen a hu- Ϫ⌬⌬Ct ⌬⌬ ϭ ⌬ Ϫ ⌬ ⌬ ϭ 2 , where Ct ( Ct(silencing)) ( Ct(non-silencing)) and Ct man thymus library for proteins that bind to KLF13. Approxi- Ϫ 6 (Ct(sample) Ct(Gus)). Values represent the fold change of target gene mately 2 ϫ 10 transformants were screened with a pSOS-KLF13 expression from PRP4 siRNA-transfected vs nonsilencing siRNA-trans- construct, and 19 putative positive clones were identified. Com- fected primary T lymphocytes. parison of cDNA sequences of the clones with the GenBank da- ELISA tabase revealed that one of them is the human homolog of the yeast CCL5 protein levels were measured in the culture supernatant using the serine-threonine kinase PRP4. To further confirm the physical in- Endogen Human RANTES ELISA Kit (Pierce) according to the manufac- teraction between KLF13 and PRP4, reciprocal coimmunoprecipi- turer’s instruction. tation experiments were performed. COS7 cells were cotransfected 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 ϳ20-fold. PRP4 mRNA decreased on day 5 and, by day 7, PRP4 mRNA dropped to the same level seen on day 1. PRP4- specific 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. FIGURE 4. PRP4 and KLF13 synergize to regulate CCL5 transcription. pGL3-RP-luc and pRL-TK were cotransfected with pcDNA3.1-KLF13 PRP4 phosphorylates KLF13, decreasing its binding affinity to (KLF13) and/or pME-HA-PRP4 (PRP4) into COS7 cells. Results are the CCL5 promoter A/B site shown as relative luciferase activity with the basal luciferase activity (vector) set to 1. The data are expressed as the average Ϯ SD from three KLF13 is the major transcription factor regulating CCL5 expres- Downloaded from independent experiments. The p values compared with vector control are sion in T lymphocytes (27, 28). KLF13 is expressed late (days indicated. 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 with pME-HA-PRP4 construct and pcDNA3.1-KLF13. Replace- tested whether PRP4 and NLK, another MAPK family member, ment of one expression plasmid with its corresponding empty vec- could phosphorylate KLF13 (Fig. 3A). The immunoprecipitants http://www.jimmunol.org/ tor was used to show specificity of coimmunoprecipitation. Only derived from PRP4- or NLK-overexpressing COS7 cells were used HA-PRP4-transfected COS7 lysates immunoprecipitated with an to phosphorylate recombinant KLF13 in a kinase assay, with im- anti-HA Ab against tagged PRP4 pulled down KLF13 (Fig. 1A), munoprecipitants from corresponding vector-transfected cells used while the reciprocal experiment using a KLF13 Ab pulled down as a negative control. Under these conditions, immunoprecipitated PRP4 (Fig. 1B). These data confirm that KLF13 binds PRP4. PRP4 phosphorylated both itself and KLF13 (Fig. 3A, lane 4). In contrast, immunoprecipitated NLK phosphorylated itself, but not PRP4, a MAPK, is expressed in T lymphocytes KLF13 (Fig. 3A, lane 2). We previously found that NLK was T lymphocytes were isolated from peripheral blood and activated recruited to the CCL5 promoter by KLF13 and subsequently phos-

with the mitogen PHA. PRP4 mRNA and protein were measured phorylates serine 10 of histone H3 upon T lymphocyte activation by guest on September 28, 2021 in resting T lymphocytes and through 7 days after activation. Low (28). These results suggest that PRP4 and NLK regulate CCL5

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 ϩ HA- PRP4). 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, anti- HA Ab, and Hoechst stained images. Arrow, Cell expressing only GFP- KLF13 that shows a different pattern of cellular distribution compared with other cells in this field that coexpress both GFP-KLF13 and HA-PRP4. The Journal of Immunology 7085 expression by phosphorylating different target proteins. Immuno- precipitated 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 immu- noprecipitant 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 su- pershifted upon the addition of anti-KLF13 Ab (Fig. 3B), but Downloaded from not with anti-p50 Ab (data not shown), indicating the specificity of binding. In the presence of PRP4, there is a ϳ50% 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 nonphos-

phorylated KLF13. http://www.jimmunol.org/ 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 induc- FIGURE 6. Knock-down of PRP4 reduces CCL5 expression in T lym- tion ( p Ͻ 0.0001) in CCL5 reporter gene activity, while expres- phocytes. A, Levels of CCL5, PRP4, and NLK mRNA after nucleofection sion of PRP4 caused a 1.4-fold increase ( p ϭ 0.01). However, of nonsilencing and PRP4 (1 and 2) siRNAs into resting human T lym- by guest on September 28, 2021 Ϯ coexpression of KLF13 and PRP4 resulted in a 5.2-fold increase phocytes followed by 30 h of PHA activation. Average SD of three ( p Ͻ 0.0001) in CCL5 promoter activity. Of note, we detected experiments are shown. B, Protein levels of PRP4 and CCL5 in human T lymphocytes after treatment with nonsilencing or PRP4 siRNAs. Left, endogenous PRP4 in COS7 cells (data not shown), explaining the Western blot analysis of PRP4 protein. PRP4-specific antisera were used to modest increase in CCL5 promoter activity in the presence of determine the amounts of protein, whereas ␣-actinin Ab was used to nor- transfected PRP4. These results indicate that PRP4 synergizes with malize for protein loading. The graph shows the ratio of PRP4:␣-actinin KLF13 to regulate CCL5 expression in vitro. expression. Right: CCL5 in culture supernatants measured by ELISA. Data represent the mean Ϯ SD of three triplicates. 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 expressed PRP4 is predominantly expressed in the nucleus of the was assessed. COS7 cells were transiently transfected with expres- cell, regardless of the presence or absence of overexpressed sion vectors corresponding to empty vector, GFP-KLF13, HA- KLF13 (Fig. 5, HA-PRP4/␣HAAb). Frames of Fig. 5 in each row PRP4, or a combination of GFP-KLF13 and HA-PRP4. No signal shows nuclei visualized by Hoechst staining. Composite was gen- was detected when cells were transfected with pcDNA3.1, and erated by merging images from frames 1–3 (Fig. 5) in each row to only a small amount of background signal was observed through- better visualize colocalization. out the cell following transfection of the pME-HA vector (Fig. 5). In the absence of PRP4, overexpressed KLF13 protein was de- Knock-down of PRP4 decreases CCL5 expression in human T tected in the nuclear compartment of all KLF13-transfected COS7 lymphocytes cells, while 45–50% of these cells also had KLF13 in the cyto- To confirm the role of PRP4 in regulating CCL5 expression in T plasm (Fig. 5, GFP-KLF13/␣-GFPAb). In contrast, when KLF13 lymphocytes, two sets of PRP4-specific siRNAs were nucleofected was coexpressed with PRP4, KLF13 resided almost exclusively in into resting human T lymphocytes. The cells were then stimulated the nucleus, indicating that PRP4 regulates the nuclear transloca- with PHA, and the expression of CCL5 was measured after 30 h tion of KLF13 (Fig. 5, GFP-KLF13 ϩ HA-PRP4/␣-GFPAb). The using RT-qPCR and ELISA. PRP4 mRNA (Fig. 6A) and protein arrows in the frames transfected with GFP-KLF13 ϩ HA-PRP4 (Fig. 6B) were suppressed Ͼ65% by both sets of siRNA. In con- (Fig. 5) mark a single cell that expresses KLF13, but not PRP4. It trast, PRP4-specific siRNAs caused only 10–15% suppression of is evident that, in the absence of PRP4, overexpressed KLF13 is NLK mRNA expression (Fig. 6A), demonstrating the specificity of distributed both in the nuclei and cytoplasm of the cell. In contrast, the siRNAs. Moreover, both sets of PRP4 siRNA repressed CCL5 overexpressed KLF13 resides exclusively in the nuclei of the other expression, with ϳ55–60% reduction in mRNA transcription and cells in the same field that is coexpressing both KLF13 and PRP4, 30–35% reduction in protein expression (Fig. 6). Thus, PRP4 reg- supporting the role of PRP4 in KLF13 nuclear translocation. Over- ulates CCL5 expression in T lymphocytes. 7086 PRP4 REGULATES CCL5 TRANSCRIPTION

Discussion is unable to phosphorylate KLF13, did not enhance transcription KLF13 is the major transcription factor that positively regulates (data not shown). This indicates that the N-terminal region of CCL5 expression in activated T lymphocytes (27). We previously PRP4 is not only important for the phosphorylation of KLF13, but reported that KLF13 protein is expressed in the adult spleen and also plays a role in regulating CCL5 expression. The apparent lung, but not in liver, brain, kidney, heart, or reproductive organs, dichotomy between decreased KLF13 DNA binding induced by and showed that KLF13 is translationally regulated through its PRP4-mediated phosphorylation and increased CCL5 expression 5Ј-untranslated region (29). In addition, KLF13 is highly phos- remains unclear, but may be related to the complexity of the CCL5 phorylated in activated T lymphocytes, suggesting that its activity enhancesome (28). In this regard, Song et al. (40) reported that the is regulated by posttranslational modification. In this study, we transcriptional coactivators p300 CREB-binding protein (CBP) show that PRP4, a MAPK family member, phosphorylates KLF13 and p300-CBP-associated factor (PCAF) act cooperatively in stim- and plays an important role in the regulation of CCL5 expression ulating KLF13 transcriptional activity. Nevertheless, p300/CBP in human T lymphocytes. and PCAF acetylate specific lysine residues in the zinc finger PRP4, originally isolated from the fission yeast Schizosaccha- DNA-binding domain of KLF13 which disrupt KLF13 DNA bind- romyces pombe, is involved in pre-mRNA splicing (33, 34). The ing. In comparison, we found that the mutation of two amino acids human homolog of PRP4 is ubiquitously expressed in multiple in the same zinc finger DNA-binding domain of KLF13 causes a tissues (31), including spleen and lung, which also express KLF13 dramatic increase in CCL5 reporter gene activity, although the (27). In addition, in T lymphocytes, the expression patterns of mutated KLF13 bound to the CCL5 A/B site with much lower affinity (data not shown). Moreover, we recently confirmed that PRP4 and KLF13 (28) mirror each other with resting cells having KLF13 recruits p300/CBP and PCAF to the CCL5 promoter in Downloaded from very low or undetectable levels of these proteins until 3 days after activated T lymphocytes (28). Studies are currently underway to activation, when both proteins are significantly induced. PRP4 be- determine whether PRP4 mediates changes in the acetylation states longs to a family of serine/arginine-rich protein-specific kinases of KLF13 in concert with other acetyltransferase proteins, such as that recognize serine-arginine-rich substrates (34). The catalytic p300/CBP and PCAF and, therefore, reduces its DNA-binding af- domain of PRP4 shows significant similarity to the JNK/stress- finity. We hypothesize that the phosphorylation of KLF13 by activated protein kinase type of MAPK including the TPY motif,

PRP4 may facilitate the assembly or disassembly of the multipro- http://www.jimmunol.org/ suggesting that PRP4 may play an important role in cell differen- tein complex at the CCL5 promoter, enabling PRP4 to further tiation (35). PRP4 has also been reported to mediate cellular sig- modify transcriptional coactivators or repressors by phosphoryla- naling (36). The N terminus of PRP4 interacts with proteins in- tion, as many ERK/MAPK and cyclic-dependent kinases have volved in splicing and nuclear hormone-regulated chromatin been shown to associate with and phosphorylate transcription fac- remodeling (37). Recently, Bennett et al. (38) reported that the C tors or transcriptional coactivators (41). terminus of PRP4 interacts with HIV-2 Gag, although HIV-2 Gag Control of CCL5 expression in T lymphocytes is complex. polyprotein is not phosphorylated by PRP4. In our study, a yeast CCL5 transcription is controlled by an enhancesome composed of two-hybrid screen demonstrated that PRP4 interacts with KLF13. different factors at various times after T lymphocyte activation We confirmed this interaction by reciprocal coimmunoprecipita- (28). KLF13, a sequence-specific DNA transcription factor, coor- by guest on September 28, 2021 tion experiments, because interactions detectable in multiple bind- dinates the induction of CCL5 expression in T lymphocytes by ing assays are unlikely to be experimental false positives (39). In ordered recruitment of proteins to the CCL5 promoter, including addition, we also demonstrated that KLF13 is a substrate of PRP4 Brahma-related gene 1 (Brg-1) (28). Brg-1 is an ATPase subunit of in kinase assays using PRP4 immunoprecipitated from transfected the SWI-SNF chromatin-remodeling complexes (42). It is re- COS7 cells. However, PRP4 (499–1007), which retains its kinase cruited to chromatin by direct interactions with DNA-binding pro- domain, showed autophosphorylation, but completely lost its abil- teins (43). Interestingly, Dellaire et al. (37) demonstrated that ity to phosphorylate KLF13 (data not shown), indicating that the Brg-1 interacts with the hypophosphorylated form of PRP4 in a truncated region of PRP4 is required for the interaction and/or transcription-dependent manner and appears to be a PRP4 sub- phosphorylation of KLF13. These findings are consistent with the strate in vitro. Nuclear receptor corepressor, a component of the previous results of others, showing that PRP4 is capable of the nuclear hormone corepressor complex, also interacts in vivo with phosphorylation of both transcription factors and serine/arginine- human PRP4. In summary, PRP4 binds to and phosphorylates rich splicing factors (36). KLF13, enhancing CCL5 expression. Therefore, PRP4 is an im- KLF13 contains multiple potential phosphorylation sites. At portant component of the enhancesome assembling over time at least 18 putative serine phosphorylation sites and 3 threonine phos- the CCL5 promoter in activated T lymphocytes. phorylation sites are predicted by NetPhos2.0 Server. Whether one or more of these sites is the PRP4 recognition sequence has not yet been determined. Although we have identified several serine phos- Disclosures phorylation sites between aa 264–88 in the C-terminal end of The authors have no financial conflict of interest. KLF13 (data not shown), deletion of this region does not completely abrogate the phosphorylation by PRP4, suggesting either References that these sites are targets of other protein kinases or that multiple 1. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and PRP4 phosphorylation sites work in concert. their role in immunity. Immunity 12: 121–127. 2. Keane, M. P., and R. M. Strieter. 1999. The role of CXC chemokines in the KLF13 binds to the A/B region of the CCL5 promoter in a regulation of angiogenesis. Chem. Immunol. 72: 86–101. dose-dependent manner (27). Although phosphorylation of KLF13 3. Campbell, J. J., and E. C. Butcher. 2000. Chemokines in tissue-specific and by PRP4 results in a decreased affinity of KLF13 for the A/B site microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12: 336–341. as demonstrated by EMSA, reporter gene assays using a CCL5 4. Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, promoter luciferase reporter indicate that coexpression of KLF13 K. Matsushima, L. H. Miller, J. J. Oppenheim, and C. A. Power. 2000. Interna- and PRP4 results in increased CCL5 promoter activity relative to tional union of pharmacology: XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 52: 145–176. transactivation by KLF13 alone. In contrast, coexpression of 5. Rossi, D., and A. Zlotnik. 2000. The biology of chemokines and their receptors. KLF13 and PRP4 (499–1007), which retains its kinase domain but Annu. Rev. Immunol. 18: 217–242. The Journal of Immunology 7087

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