G-protein-independent Activation of Tyk2 by the Platelet-activating Factor Receptor
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m100720200
ISSN1083-351X
AutoresViktoria Lukashova, Claude Asselin, John J. Krolewski, Marek Rola‐Pleszczynski, Jana Staňková,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoPlatelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activity through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed that PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the monocytic cell lines U937 and MonoMac-1. PAF-initiated Tyk2 phosphorylation was also observed in COS-7 cells transiently transfected with the human PAF receptor (PAFR) and Tyk2 cDNAs. In addition, we found that Tyk2 co-immunoprecipitated and co-localized with PAFR, independently of ligand binding. Deletion mutants of Tyk2 indicated that the N terminus of the kinase was important for the binding to PAFR. Activation of Tyk2 was followed by a time-dependent 2–4-fold increase in the level of tyrosine phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 and a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In MonoMac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stimulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cells were transfected with PAFR and constructs containing PAFR promoter 1, coupled to the luciferase reporter gene, PAF induced a 3.6-fold increase in promoter activation in the presence of Tyk2. Finally, PAFR mutants that could not couple to G-proteins were found to effectively mediate Tyk2 activation and signaling. Taken together, these findings suggest an important role for the Janus kinase/STAT pathway in PAFR signaling, independent of G-proteins, and in the regulation of PAF receptor expression by its ligand. Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activity through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed that PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the monocytic cell lines U937 and MonoMac-1. PAF-initiated Tyk2 phosphorylation was also observed in COS-7 cells transiently transfected with the human PAF receptor (PAFR) and Tyk2 cDNAs. In addition, we found that Tyk2 co-immunoprecipitated and co-localized with PAFR, independently of ligand binding. Deletion mutants of Tyk2 indicated that the N terminus of the kinase was important for the binding to PAFR. Activation of Tyk2 was followed by a time-dependent 2–4-fold increase in the level of tyrosine phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 and a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In MonoMac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stimulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cells were transfected with PAFR and constructs containing PAFR promoter 1, coupled to the luciferase reporter gene, PAF induced a 3.6-fold increase in promoter activation in the presence of Tyk2. Finally, PAFR mutants that could not couple to G-proteins were found to effectively mediate Tyk2 activation and signaling. Taken together, these findings suggest an important role for the Janus kinase/STAT pathway in PAFR signaling, independent of G-proteins, and in the regulation of PAF receptor expression by its ligand. GTP-binding regulatory protein G-protein-coupled receptor Janus kinase pertussis toxin platelet-activating factor platelet-activating factor receptor signal transducers and activators of transcription antibody base pair(s) phospholipid interleukin tumor necrosis factor Platelet-activating factor (PAF)1 is a potent phospholipid mediator involved in a variety of biological processes. PAF plays a role in allergic disorders and inflammation, and it has also been implicated in reproductive, cardiovascular, neurological, and immune systems. PAF is produced by many cells, including monocytes, endothelial cells, neutrophils, and lymphocytes. Many of these cell types can themselves become targets of PAF bioactions (1Braquet P. Touqui L. Shen T.Y. Vargaftig B.B. Pharmacol. Rev. 1987; 39: 97-145PubMed Google Scholar, 2Snyder F. Am. J. Physiol. 1990; 259: C697-C708Crossref PubMed Google Scholar, 3Venable M.E. Zimmermann G.A. McIntyre T.M. Prescott S.M. J. Lipid Res. 1993; 34: 691-702Abstract Full Text PDF PubMed Google Scholar). PAF mediates its effects via the activation of a seven-transmembrane domain, G-protein-coupled receptor (GPCR). Upon binding to its receptor, PAF simulates a number of signal transduction pathways. These include phospholipid turnover, through phospholipase Cγ (PLCγ) and PLCβ activation, in many systems, including platelets, macrophages, B cell lines, endothelial cells, and Kupffer cells (4Izumi T. Shimuzu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar, 5Chao W. Olson M.S. Biochem. J. 1993; 292: 617-629Crossref PubMed Scopus (418) Google Scholar, 6Shukla S.D. Lipids. 1991; 26: 1028-1033Crossref PubMed Scopus (41) Google Scholar). PAF also activates PLA2, PLD, and phosphatidylinositol 3-kinase in many different cells and tissues (reviewed in Ref. 4Izumi T. Shimuzu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar). Previous studies showed that PAF induces tyrosine phosphorylation of numerous cellular proteins, such as p125fak in human endothelial cells and brain (7Soldi R. Sanavio F. Aglietta M. Primo L. Defilippi P. Marchisio P.C. Bussolino F. Oncogene. 1996; 13: 515-525PubMed Google Scholar, 8Calcerrada M.C. Calatan R.E. Pérez-Alvarez M. Miguel B.G. Martinez A.M. Brain Res. 1999; 835: 275-281Crossref PubMed Scopus (9) Google Scholar) and pp60c-src (9Dhar A. Shukla S.D. J. Biol. Chem. 1994; 269: 9123-9127Abstract Full Text PDF PubMed Google Scholar), PLCγ, Fyn, Syk, Lyn, and p85 regulatory subunit of phosphatidylinositol 3-kinase in human B cell lines (4Izumi T. Shimuzu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar, 10Kuruvilla A. Pielop C. Shearer W.T. J. Immunol. 1994; 153: 5433-5442PubMed Google Scholar, 11Huang S.J. Monk P.N. Downes C.P. Becker E.L. Sha'afi R.I. Biochem. J. 1988; 249: 839-845Crossref PubMed Scopus (49) Google Scholar). Recent data indicate that a protein tyrosine kinase pathway may also be implicated in PAF-induced activation of PLD, mitogen-activated protein kinase and PLA2, but the nature of the kinases involved and the molecular mechanisms through which PAF receptor activates tyrosine kinase signals are not well understood (4Izumi T. Shimuzu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar). The involvement of different subtypes of G-proteins has been documented for some signaling pathways. PAFR couples to both pertussis toxin (PTX)-sensitive and PTX-insensitive G-proteins, and thus PAF-induced activation of PLC isoforms or other enzymes could vary depending on cell type. For example, in macrophages and neutrophils, phosphoinositol turnover induced by PAF is sensitive to PTX and might be attributed to G-protein Giα2 and Giα3 subunits expressed in these cells (4Izumi T. Shimuzu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar,12Rezaul K. Sada K. Inazu T. Yamamura H. Biochem. Biophys. Res. Commun. 1997; 239: 23-27Crossref PubMed Scopus (9) Google Scholar), whereas in Chinese hamster ovary cells, mitogen-activated protein kinase activation in response to PAF is mediated by Gαo (13Van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). PAF has been shown to induce the expression of early response genes such as c-fos and c-jun in many cells and tissues, as well as regulating the transcription of cytokine genes such as IL-6, IL-1, IL-8, and TNF-α in human monocytes, TNF in NK cells, and IL-6 in endothelial cells and neutrophils (reviewed in Ref.14Rola-Pleszczynski M. Thivierge M. Gagnon M. Lacasse N. Stankova J. J. Lipid Mediators. 1993; 6: 175-181PubMed Google Scholar). PAF also up-regulates the expression of its own receptor in several cell types (15Mutoh H. Ishii S. Izumi T. Kato S. Shimizu T. Biochem. Biophys. Res. Commun. 1994; 205: 1137-1142Crossref PubMed Scopus (57) Google Scholar), including human alveolar macrophages (16Shimizu T. Mutoh H. Kato S. Adv. Exp. Med. Biol. 1996; 416: 79-84Crossref PubMed Google Scholar) and rat epithelial cells (17Wang H. Tan X. Chang H. Huang W. Gonzalez-Crussi F. Hsueh W. Immunology. 1999; 97: 447-454Crossref PubMed Scopus (18) Google Scholar), thus potentially providing a positive feedback loop for PAF action. Two distinct promoters are involved in the transcriptional regulation of PAFR gene expression in various cell types, resulting in two transcripts, 1 and 2, which differ only in their untranslated region. The human PAFR transcript 1 is ubiquitous but is most abundant in peripheral leukocytes and may be chiefly regulated by inflammatory and various pathological processes (18Mutoh H. Bito H. Minami M. Nakamura M. Honda Z. Izumi T. Nakata R. Kurachi Y. Terano A. Shimizu T. FEBS Lett. 1993; 322: 129-134Crossref PubMed Scopus (95) Google Scholar). The human PAFR transcript 2 is found in the heart, lung, spleen, and kidney and can be regulated by retinoic acid, thyroid hormone T3, estrogen, and transforming growth factor-β, among others (19Mutoh H. Kume K. Sato S. Kato S. Shimizu T. Biochem. Biophys. Res. 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Commun. 1993; 197: 1443-1449Crossref PubMed Scopus (16) Google Scholar). The nature of the transcription factors involved in PAF-induced human PAFR gene expression has yet to be identified. It has been shown that PAF can induce NF-κB activation and receptor transcription through three NF-κB consensus sites in promoter 1 upstream of –459 bp in certain cell types (19Mutoh H. Kume K. Sato S. Kato S. Shimizu T. Biochem. Biophys. Res. Commun. 1994; 205: 1130-1136Crossref PubMed Scopus (30) Google Scholar, 26Kravcenko V.V. Pan Z. Han J. Herbert J.-M. Ulevitch R.J. Ye R.D. J. Biol. Chem. 1995; 270: 14928-14934Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway is one of the major mechanisms by which cytokine receptors transduce intracellular signals. To date, four mammalian Jaks have been identified (Jak1, Jak2, Jak3, and Tyk2), and seven STATs have been characterized (27Pellegrini S. 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In the present report, we show that PAF-mediated PAFR transcription is dependent on the activation of the Jak/STAT pathway. The Tyk2 kinase is activated in response to PAF in myeloid cells and in a transfected cell system. This activation leads to subsequent tyrosine phosphorylation of STATs 1, 2, 3, and 5. Tyk2 associates with PAFR, independently of agonist binding, and its presence is obligatory for human PAFR promoter 1 activation. Finally, Tyk2 activation by PAF is independent of G-protein coupling. PAF was from the Cayman Chemical Co. (Ann Arbor, MI). WEB 2086, a PAF-specific antagonist was from Roche Molecular Biochemicals. Antibodies used were rabbit polyclonal anti-Tyk2 and anti-STATs (Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti-N-terminal region of Tyk2 (Transduction Laboratories, Missisavga, Canada); monoclonal anti-HA (anti-hemagglutinin; BAbCO, Richmond, CA), monoclonal anti-Tyr(P) was a kind gift from Drs. P. Angelisova and V. Horejsi (Institute of Molecular Genetics, Prague, Czech Republic), and monoclonal anti-c-Myc was form ATCC (Manassas, VA). Nonpertinent control antibodies OKT3 and polyclonal anti-LTB4 receptor were from ATCC and Cayman Chemical Co., respectively. Horseradish peroxidase-conjugated goat anti-mouse and donkey anti-rabbit antibodies were from Amersham Pharmacia Biotech. Human Tyk2 wild type and mutant cDNAs were generated as described (33Yan H. Piazza F. Krishnan K. Pine R. Krolewski J.J. J. Biol. Chem. 1998; 273: 4046-4051Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The human PAFR cDNA was a gift form Dr. R. Ye (University of Chicago, Chicago, IL). Murine STAT1α and STAT3 cDNAs were kindly provided by Dr. J. E. Darnell (Rockefeller University, New York, NY) and Dr. S. Akira (Hyogo College of Medicine, Japan), respectively. MonoMac-1 (DSM ACC252) and U937 (ATCC CRL-1593.2) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and the ATCC, respectively. COS-7 were kindly provided by Dr. G. Guillemette (Université de Sherbrooke, Québec, Canada). FuGENETM6 transfection reagent was from Roche Molecular Biochemicals. pJ3M expression vector was a gift from Dr. J. Chernoff (Fox Chase Cancer Center) COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. Cells were plated in 100-mm dishes (1 × 106cells/dish) and transiently transfected with human PAFR cDNA cloned into the pJ3M expression vector with or without hTyk2 or pcDNA3 cDNA (4–7 μg of DNA, total) using 8–14 μl of FuGENE. 48 h after transfection, cells were incubated without serum for 1 h and then stimulated with PAF (10−7m) or left unstimulated. Cells were lysed, and extracts used as indicated below for immunoprecipitation. U937 were maintained in RPMI medium supplemented with 10 mmHEPES, 10% fetal bovine serum, 100 units/ml ampicillin, 100 μg/ml streptomycin. In the case of MonoMac-1 cells, 10 mmnonessential amino acids and 10 mm sodium pyruvate were added. U937 or MonoMac-1 cells (20 × 106) were incubated in medium without fetal bovine serum 24 h, stimulated with PAF (10−8m) for the indicated times, and lysed in buffer: 50 mm Tris, pH 7.5, 1 mmEGTA, 150 mm NaCl, 1 mm NaF, 1 mmNa3VO4, 1% Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, 0.25% sodium deoxycholate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin, 30 min on ice. Lysates were precleared with 25 μg of protein A-Sepharose for 30 min and incubated with anti-Tyk2 or anti-STAT Abs overnight at 4 °C. Proteins of interest were precipitated by incubation with 100 μg of protein A-Sepharose for 2 h at 4 °C. In case of immunoprecipitation of PAFR with c-Myc Ab, protein A-Sepharose was gently shaken in lysis buffer containing 1% bovine serum albumin for 30 min at room temperature before use. After washing four times in 0.5× lysis buffer, complexes were dissolved in 1× loading buffer, separated by 8% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Membranes were blocked in Tris-buffered saline with 2.5% gelatin for 1 h and incubated with anti-Tyr(P) in Tris-buffered saline-Tween 0.1% + 0.5% gelatin overnight at 4 °C. After washing and incubation with secondary antibodies, an enhanced chemiluminescent detection system was used for protein detection (Amersham Pharmacia Biotech). Membranes were stripped by incubation in 62.5 mm Tris-HCl, pH 6.8, 2% SDS, and 10 mm 2-mercaptoethanol for 30 min at 50 °C. After washing, membranes were reprobed with appropriate Ab and developed as described above. To confirm receptor specificity, cells were pretreated with WEB 2086 (10−5–10−7m) for 20 min at 37 °C before PAF stimulation. Transiently transfected COS-7 cells (1–1.6 μg of cDNA, 2 × 105 cells) were plated on 25-mm coverslips and processed as previously described (16Shimizu T. Mutoh H. Kato S. Adv. Exp. Med. Biol. 1996; 416: 79-84Crossref PubMed Google Scholar), with modifications. For the analysis of PAFR and Tyk2 colocalization, cells were fixed in 4% paraformaldehyde (Sigma) and permeabilized in 0.1% saponin (Sigma). During all subsequent washing procedures, 0.01% saponin was added. For STAT translocation experiments, MonoMac-1 cells were plated on poly-l-lysine-coated coverslips and preincubated 1 h with sodium orthovanadate (60 μm, Sigma) before PAF stimulation. In these experiments, 0.1% Triton was used for cellular permeabilization. The cells were incubated with anti-c-Myc or isotype control OKT3 or nonpertinent anti-BLTR Abs. Cells were examined with a scanning confocal microscope (NORAN Instruments Inc., Middleton, WI) equipped with a krypton/argon laser and coupled to an inverted microscope with a 40× oil immersion objective (Nikon). Specimens were excited at 488 and 568 nm. Emitted fluorescein isothiocyanate fluorescence and rhodamine fluorescence were measured at wavelengths 525–550 and 590 nm, respectively. Optical sections were collected at 1-μm intervals with a 10-μm pinhole aperture. Digitized images were obtained with 256 times line averaging and enhanced with Intervision software (NORAN Instruments Inc.) on a Silicon Graphics O2-work station. The promoterless luciferase reporter gene plasmid, pGL3basic (Promega), was used to construct all PAFR promoter reporter plasmids. For the p.98Luc construct, polymerase chain reaction was used to amplify the desired 980-bp fragment (–980 to +37) of the human leukocyte promoter 1 of PAFR from the Raji cell line DNA, by using forward (5′-CGTGCGCACAGGTCTTATATCTTGTAAGATGACT-3′) and reverse (5′-GTCTGTCGTATCTCCGACTCCGACCTTCGAACG-3′) primers. The p0.16Luc construct (PAFR promoter sequence from –157 to +37) was prepared by MluI-AccI digestion of the p0.98Luc and treating the product with the Klenow fragment of DNA polymerase I before self-ligation. Cells were transfected in 24-well plates with a total of 0.4 μg of cDNAs. 48 h after transfection, cells were harvested and assayed for luciferase activity. In the present study, we investigated whether the Jak/STAT signal transduction pathway was activated upon PAF stimulation of human monocytic cell lines U937 and MonoMac-1. Cells were stimulated with PAF for 1 min, in the absence or presence of WEB 2086, a PAF-specific antagonist. Cellular extracts were then immunoprecipitated with anti-Tyk2 antibodies and revealed with an anti-phosphotyrosine Ab. As illustrated in Fig. 1, Tyk2 exhibited a basal level of tyrosine phosphorylation even in quiescent MonoMac (Fig.1 A) and U937 (Fig. 1 B) cells. PAF induced a significant increase in Tyk2 tyrosine phosphorylation after 1 min, and this increase was completely blocked by WEB 2086 in both MonoMac and U937 cells, indicating a receptor mediated signaling. In kinetic studies shown in Fig. 2, A andB, PAF promoted a rapid (within 1–2 min) and transient increase in tyrosine phosphorylation of Tyk2 in MonoMac cells. Equivalent amounts of Tyk2 proteins were present in immunoprecipitates, as detected by reblotting of membranes with anti-Tyk2 antibody. Jak2 was also phosphorylated in response to PAF but not Jak1 or Jak3 (results not illustrated). Next, we examined whether Tyk2 could be activated in a reconstructed system. COS-7 cells were transiently transfected with PAFR and Tyk2 cDNA. As shown in Fig. 2,C and D, PAF also stimulated an increase in the level of tyrosine phosphorylation of Tyk2 in cells cotransfected with PAFR and Tyk2. This increase was reproducible, but at lower levels than the increase in the myeloid cells, possibly due to the high basal phosphorylation levels of the transfected Tyk2. Interestingly, when Jak2 was co-transfected with Tyk2 and PAFR, Tyk2 phosphorylation achieved the same levels as in MonoMac cells (results not shown).Figure 2Tyk2 tyrosine phosphorylation induced by PAF. A, MonoMac-1 cells were stimulated with PAF (10−8m) for the indicated times. Tyk2 tyrosine phosphorylation was assessed by immunoprecipitation and Western blotting as in Fig. 1. B, densitometry ratios of pTyk2/Tyk2 are shown as fold induction, the ratio for unstimulated cells (NS) being set at 1. Results are mean of three independent experiments ± S.E. C, COS-7 cells were transiently transfected with PAFR and Tyk2, and 48 h after transfection, cells were stimulated with PAF for the indicated times. Tyk2 tyrosine phosphorylation was assessed by immunoprecipitation and Western blotting as in Fig. 1. D, densitometry ratios of pTyk2/Tyk2 are shown as fold induction, the ratio for unstimulated cells (NS) being set at 1. Results shown are representative of five independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interaction between PAFR and Tyk2 was first examined by confocal microscopy. As illustrated in Fig.3 A, in MonoMac cells, both the PAFR (top panels, green) and Tyk2 (center panels, red) had a predominantly plasma membrane distribution. In transfected Cos-7 cells, c-Myc-tagged PAF receptors (Fig. 3 B, top panels) were localized in the plasma membrane, whereas Tyk2 (red staining) was mainly distributed in intracellular compartments, with some clusters found at the cellular membrane in unstimulated cells (Fig. 3 A, middle panels, NS). When the two labels were overlaid, the yellow areas indicate the co-localization of PAFR and Tyk2. The majority of the colocalized proteins was found in the plasma membrane regions (Fig. 3, Aand B, bottom panels), and PAF stimulation did not change either the localization of Tyk2 or the intensity of the co-localization (Fig. 3, A and B, PAF), indicating that PAF stimulation did not induce a redistribution of Tyk2. To confirm that Tyk2 could associate with PAFR, lysates from transiently cotransfected COS-7 cells with c-Myc-tagged PAFR and Tyk2 cDNAs, unstimulated or stimulated with PAF, were immunoprecipitated with anti-c-Myc antibodies. Protein complexes were analyzed by Western blotting with anti-Tyk2 Abs. Data shown in Fig. 3 C indicate that in both unstimulated and stimulated cells, Tyk2 coprecipitated with PAFR (top panel). This finding suggests that Tyk2 constitutively associates with the receptor independently of ligand binding. No difference was observed in the amount of PAFR in each lane (Fig. 3 C, bottom panel) when the membrane was reblotted with an anti-c-Myc Ab. Under the same conditions, we were unable to immunoprecipitate Jak2 with the PAFR, before or after PAF stimulation (results not shown). To determine which region of Tyk2 bound PAFR, we used truncation mutants of the kinase. COS-7 cells were transfected with PAFR and Tyk2 (WT and mutants). Fig.4 shows that mutants containing all or parts of the N terminus could immunoprecipitate with the PAFR. The membrane was first immunoblotted with an Ab that recognizes the N terminus of Tyk2 (Fig. 4, top panel) and then stripped, and an anti-HA Ab was used to reveal the HA tag incorporated into the 263–601 construct, because the epitope recognized by the anti-Tyk2 Ab had been deleted in this mutant (middle panel). Thebottom panel shows that equivalent amounts of PAFR had been immunoprecipitated. Activation of the Jak family of kinases by cytokine receptors is followed by tyrosine phosphorylation of the STAT family of transcription factors. In order to determine whether PAFR signaling also involved the STATs, these proteins were immunoprecipitated from unstimulated and PAF-stimulated MonoMac-1 cells. As shown in Fig.5, PAF induced a time-dependent, transient, 2–4-fold increase in the level of tyrosine phosphorylation of STAT1α and STAT2 and a sustained 2–6-fold increase in STAT5 tyrosine phosphorylation, as determined by densitometric analysis. Weak activation of STAT3 (1.3–2-fold) was detected in PAF treated cells (Fig.6 C), but no PAF-induced STAT4 or STAT6 tyrosine phosphorylation was observed (data not shown). Interestingly, STAT5 tyrosine phosphorylation persisted on a relatively high level over the time period tested (1 h), whereas for STAT1α and STAT2, a peak of phosphorylation was observed at 5–15 min, which declined to basal level by 60 min.Figure 6Nuclear translocation of STAT1 and STAT3 in response to PAF. A, COS-7 cells transfected with c-Myc-tagged PAFR, Tyk2, and STAT1α or STAT3 cDNAs were analyzed by confocal microscopy prior to (left) (NS) or 30 min following (right) treatment with PAF (10−7m). Images in all panels are of different cells. PAFR was visualized with anti-c-Myc Ab and fluorescein isothiocyanate-conjugated secondary Ab (green), and STATs were detected by incubation with corresponding anti-STAT Ab and rhodamine-conjugated secondary Ab (red). B,MonoMac-1 cells were stimulated with PAF (10−8m), stained with corresponding anti-STAT Abs and fluorescein isothiocyanate-conjugated secondary Abs (green). Nuclei were stained with propidium iodide (red).C, MonoMac-1 cells were treated 30 min with 500 units of interferon-γ or 20 ng/ml of IL-6 stained with anti-STAT1 or anti-STAT3 Abs (green) and propidium iodide (red). Translocation of STATs in response to both cytokines is seen in the overlapped image (yellow).View Large Image Figure ViewerDownload Hi-res image Download (PPT) STAT phosphorylation induced by growth factors and cytokines results in the translocation of the phosphorylated STATs to the nucleus. We next sought to confirm that PAF-induced phosphorylation of STATs correlates with functional activation of these proteins. We investigated the effect of PAF on the subcellular localization of STAT1α and STAT3. We concentrated on these two STATs as they have putative binding sites in the promoter 1 of PAFR. For these experiments, we employed COS-7 cells transiently expressing PAFR, Tyk2, and STAT1α or STAT3 cDNAs (Fig. 6 A) or MonoMac-1 cells (Fig. 6, B andC). Nuclear translocation was analyzed by confocal microscopy prior to or following stimulation with PAF for 30 min. In the transfected, unstimulated cells, STAT proteins were distributed diffusely in the intracellular compartments, including the cytoplasm and nucleus (Fig. 6 A, left panels, red staining). The PAF receptor was found predominantly on the cellular membrane (Fig.6 A, left panels, green staining). However, after treatment with PAF (10−7m) for 30 min, STAT1α and STAT3 were detected predominantly in the nucleus (Fig.6 A, right panels, red staining), and the PAFR could be seen internalizing in vesicles (Fig. 6 A, right panels, green staining). STAT translocation was not observed in the absence of cotransfected Tyk2 (results not illustrated). Comparable results were obtained after PAF stimulation of MonoMac cells. Fig. 6, B and C, shows MonoMac 1 cells with STAT1 and STAT3 stained green and the nucleus stained red with propidium iodide; in unstimulated cells (NS, left panels), STAT1 protein was distributed mainly in the cytoplasm, but some was also found in the nuclear area, whereas STAT3 was distributed exclusively in the cytoplasm. After PAF stimulation (Fig. 6 B, right panels), the majority of both STAT1 and STAT3 translocated to the nucleus (yellow). Interferon-γ and IL-6 were used as controls for translocation of STAT1 and STAT3, respectively (Fig.6 C). PAF has been reported to in
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