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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 References This article cites 43 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/178/11/7081.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 coimmunopre- cipitation. 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 pur- chased from Roche Diagnostic Systems. Anti-V5 mouse mAb was pur- chased 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 trans- fected 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.
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