FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079200708> ?p ?o ?g. }

• W2079200708 endingPage "19629" @default.
• W2079200708 startingPage "19617" @default.
• W2079200708 abstract "Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells. Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells. Activation of leukocytes through multichain immunoreceptors (TCR, 3The abbreviations used are: TCR, T cell receptor; aa, amino acid; Brij-98, polyoxyethylene 20 oleyl ether; J-iPRR7, Jurkat clones with PRR7-inducible expression; LM, lauryl maltoside (n-dodecyl-β-d-maltoside); PBL, peripheral blood lymphocytes; PSD, postsynaptic density; RSL1, RheoSwitch Ligand 1, diacylhydrazine [(N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine]; SFK, Src family kinase; TRAP, transmembrane adaptor protein; PMA, phorbol 12-myristate 13-acetate; PHA, phytohemagglutinin; qPCR, quantitative PCR; Z-VAD-FMK, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone; PLC, phospholipase C. BCR, FcRs) involves a complex array of membrane-linked and cytoplasmic proteins, including transmembrane and cytoplasmic adaptor proteins (1Simeoni L. Kliche S. Lindquist J. Schraven B. Curr. Opin. Immunol. 2004; 16: 304-313Crossref PubMed Scopus (43) Google Scholar). Several of the transmembrane adaptors (TRAPs) are associated with lipid rafts, namely LAT (2Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar, 3Brdiĉka T. Cerný J. Horejŝí V. Biochem. Biophys. Res. Commun. 1998; 248: 356-360Crossref PubMed Scopus (50) Google Scholar), PAG (4Brdicka T. Pavlistová D. Leo A. Bruyns E. Korínek V. Angelisová P. Scherer J. Shevchenko A. Hilgert I. Cerný J. Drbal K. Kuramitsu Y. Kornacker B. Horejsí V. Schraven B. J. Exp. Med. 2000; 191: 1591-1604Crossref PubMed Scopus (405) Google Scholar, 5Kawabuchi M. Satomi Y. Takao T. Shimonishi Y. Nada S. Nagai K. Tarakhovsky A. Okada M. Nature. 2000; 404: 999-1003Crossref PubMed Scopus (464) Google Scholar), NTAL (6Brdicka T. Imrich M. Angelisová P. Brdicková N. Horváth O. Spicka J. Hilgert I. Lusková P. Dráber P. Novák P. Engels N. Wienands J. Simeoni L. Osterreicher J. Aguado E. Malissen M. Schraven B. Horejsí V. J. Exp. Med. 2002; 196: 1617-1626Crossref PubMed Scopus (180) Google Scholar, 7Janssen E. Zhu M. Zhang W. Koonpaew S. Zhang W. Nat. Immunol. 2003; 4: 117-123Crossref PubMed Scopus (134) Google Scholar), and LIME (8Brdicková N. Brdicka T. Angelisová P. Horváth O. Spicka J. Hilgert I. Paces J. Simeoni L. Kliche S. Merten C. Schraven B. Horejsí V. J. Exp. Med. 2003; 198: 1453-1462Crossref PubMed Scopus (89) Google Scholar, 9Hur E.M. Son M. Lee O.H. Choi Y.B. Park C. Lee H. Yun Y. J. Exp. Med. 2003; 198: 1463-1473Crossref PubMed Scopus (75) Google Scholar), whereas others (LAX, SIT, TRIM, GAPT) are present in non-raft membrane (10Zhu M. Janssen E. Leung K. Zhang W. J. Biol. Chem. 2002; 277: 46151-46158Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 11Marie-Cardine A. Kirchgessner H. Bruyns E. Shevchenko A. Mann M. Autschbach F. Ratnofsky S. Meuer S. Schraven B. J. Exp. Med. 1999; 189: 1181-1194Crossref PubMed Scopus (60) Google Scholar, 12Bruyns E. Marie-Cardine A. Kirchgessner H. Sagolla K. Shevchenko A. Mann M. Autschbach F. Bensussan A. Meuer S. Schraven B. J. Exp. Med. 1998; 188: 561-575Crossref PubMed Scopus (115) Google Scholar, 13Liu Y. Zhang W. J. Leukoc. Biol. 2008; 84: 842-851Crossref PubMed Scopus (16) Google Scholar). All of these proteins are composed of a short N-terminal extracellular peptide, a single transmembrane segment, and a cytoplasmic part containing multiple tyrosine and other protein interaction motifs. In the case of the raft-associated proteins, a palmitoylation motif (CXXC or CXC) lies between the transmembrane segment and the intracellular part (14Horejsí V. Immunol. Lett. 2004; 92: 43-49Crossref PubMed Scopus (38) Google Scholar). TRAPs play positive and negative regulatory roles in immunoreceptor signaling. The most prominent role is played by LAT, which is essential for several aspects of TCR signaling and plays an important role in the immunoreceptor signaling in other leukocyte subsets (15Horejsí V. Otáhal P. Brdicka T. FEBS J. 2010; 277: 4383-4397Crossref PubMed Scopus (7) Google Scholar). In addition, experiments on genetic models have assigned important regulatory functions to other members of TRAP family, including NTAL, LAX, TRIM, and SIT (16Fuller D.M. Zhang W. Immunol. Rev. 2009; 232: 72-83Crossref PubMed Scopus (24) Google Scholar, 17Koelsch U. Schraven B. Simeoni L. J. Immunol. 2008; 181: 5930-5939Crossref PubMed Scopus (16) Google Scholar). In an effort to discover additional TRAPs of functional importance, we performed an in silico search for proteins possessing the features characteristic of known TRAPs. Among 149 candidate genes, one of the best hits was PRR7. Previously, PRR7 was identified in the PSD fraction of rat forebrain tissue by a proteomic approach (18Murata Y. Doi T. Taniguchi H. Fujiyoshi Y. Biochem. Biophys. Res. Commun. 2005; 327: 183-191Crossref PubMed Scopus (22) Google Scholar). It was shown to associate with PSD-95 (via a PDZ domain binding motif) and was found in a protein complex containing the N-methyl-d-aspartate receptor subunits NR1 and NR2B. These biochemical data suggested that PRR7 could be potentially involved in modulation of neural activities. In the present study we found that PRR7 is weakly expressed in many other tissues, including immune cells, and is up-regulated during T cell activation. Moreover, we show that PRR7 overexpression in Jurkat cells substantially affected TCR signaling and cell survival. The in silico search in human genome (USCS build hg16) for proteins possessing the features characteristic of known TRAPs was done with following parameters: a short extracellular N-terminal sequence, a single hydrophobic sequence starting at amino acids (aa) 5–50 (prediction using TMHMM Version 2.0) (19Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9164) Google Scholar), a palmitoylation motif at aa 20–60 (CXC or CXXC), tyrosine-based phosphorylation motifs (YXX(I/L/V/A)) and/or C-terminal Group I PDZ binding motifs ((S/T)X(L/V)). RNA was isolated using a Mini RNA purification kit (Zymo Research, Orange, CA) or TRI Reagent RT (MRC, Cincinnati, OH). Contaminating DNA was removed using a DNAfree kit (Ambion, Austin, TX). A human normal tissue FirstChoice RNA Survey Panel and a lymph node FirstChoice Total RNA were purchased from Ambion. Human hippocampal RNA was purchased from BioChain Institute (Hayward, CA). Total RNA (1 μg) was transcribed using SuperScript III RT (Invitrogen) with a combination of random pentadecamer and anchored oligo(dT)20 primers. RT-qPCR was performed using a LightCycler 480 SYBR Green I Master chemistry (Roche Applied Science) and an amount of cDNA equivalent to 20 ng of total RNA. Primers specific for human PRR7 were 5′-tgtccagtgaagcgtctgag-3′ (forward) and 5′-aagcacgtgaggaacgtgta-3′ (reverse). Raw RT-qPCR data (Ct values) were preprocessed (normalization to optimal reference genes, GAPDH or β-microglobulin, relative expression calculation) and analyzed using GenEx Software (MultiD, Göteborg, Sweden). Total RNA was isolated from 1 × 107 cells using TRI Reagent RT. RNA was resolved (15 μg/sample) by formaldehyde-agarose gel electrophoresis, transferred to positively charged nylon membrane, and hybridized to radioactively ([α-32P]dCTP)-labeled probe in Super Hyb solution (MRC). The probe was prepared from cloned PRR7 cDNA using DecaLabel DNA labeling kit (Fermentas, Ontario, Canada). Cell line MEG-01 was provided by S. Watson (University of Birmingham, Birmingham, UK), MOLT-4 and HPB-ALL were from the IMG cell line collection (Institute of Molecular Genetics, Prague, Czech Republic), and Caco-2, U937, and THP-1 were from DSMZ (Braunschweig, Germany). Jurkat, Ramos, and COS-7 cell lines were from ATCC (Manassas, VA), and HEK293FT cells were obtained from Invitrogen. Caco-2, HEK293FT, and COS-7 cells were cultured in DMEM supplemented with 10% FCS (Biochrom AG, Berlin, Germany) and antibiotics at 37 °C in 5% CO2. All other cells were cultured in RPMI 1640 supplemented with 10% FCS and antibiotics at 37 °C in 5% CO2. Human peripheral blood lymphocytes (PBL) were isolated from whole blood of healthy donors by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation. For depletion of adherent cells, PBL were subjected to plastic adherence for 2 h at 37 °C. PBL were cultured as stated above for Jurkat cells. The coding region of human PRR7 was amplified from human T cell line MOLT-4 cDNA and inserted into mammalian expression plasmid pEFIRES-P, provided by Dr. S. Hobbs (Institute of Cancer Research, London, UK) (20Hobbs S. Jitrapakdee S. Wallace J.C. Biochem. Biophys. Res. Commun. 1998; 252: 368-372Crossref PubMed Scopus (222) Google Scholar). For inducible expression, PRR7 was subcloned into the pZX-LR vector in-frame with the hemagglutinin (HA) tag. The pZX-LR vector was a kind gift of Dr. Claude Labrie (CHUL Research Center, Quebec, Canada) (21Lessard J. Aicha S.B. Fournier A. Calvo E. Lavergne E. Pelletier M. Labrie C. Prostate. 2007; 67: 808-819Crossref PubMed Scopus (13) Google Scholar). Deletion mutants of PRR7 (Δ2–39, Δ44–274, Δ114–274, Δ151–274, Δ159–274, Δ171–274, Δ207–274, ΔTAV) were generated by PCR and fused to enhanced green fluorescent protein (EGFP) in the pEGFP vector (Clontech, Palo Alto, CA). A tyrosine mutant of PRR7 (all Tyr residues mutated to Phe) was generated by sequential PCR oligonucleotide-directed site-specific mutagenesis and subcloned into the pEGFP vector (Clontech). For co-transfection experiments in COS-7 cells, the following cDNA constructs were used: Src in the pSM vector provided by Dr. A. Weiss (University of California, San Francisco, CA), FLAG-tagged Lck inserted into the pcDNA3 vector provided by Dr. R. Abraham (Mayo Clinic, Rochester, MN), FynT and FynF (constitutively active Fyn) in the pEF-BOS vector provided by Dr. B. Schraven (Otto-von-Guericke-University, Magdeburg, Germany), Yes in the pSG5 vector provided by Dr. C. Benistant (CRBM, CNRS, Universities of Montpellier I and II, France), Myc-tagged Lyn in the pcDNA3.1 vector provided by Dr. S. Watson (University of Birmingham, Birmingham, UK), FLAG-tagged Hck in the pcDNA1 vector provided by Dr. G. Langsley (Institut Pasteur, Paris, France). Hybridomas producing monoclonal antibodies (mAbs, TRAP-3/01–13) to human PRR7 were prepared by standard hybridoma techniques using mice immunized with the recombinant protein corresponding to aa 147–274. Using the deletion mutants, the epitope recognized by the mAbs was mapped to the stretch of aa 147–170. The mAbs were cross-reactive with murine and rat PRR7 in immunoblots (not shown). The phosphotyrosine antibody (P-TYR-02) used for detection of phospho-LAT in Jurkat whole cell lysates was developed in our laboratory as well. For cell stimulation, the following non-commercial antibodies were used: C305 IgM mAb to Jurkat cell TCR provided by Dr. A. Weiss and 248.23.2 IgM mAb to CD28 (22Qiao L. Schürmann G. Betzler M. Meuer S.C. Gastroenterology. 1991; 101: 1529-1536Abstract Full Text PDF PubMed Scopus (87) Google Scholar). Antibodies to the following antigens were obtained from the indicated commercial sources: LAT (LAT-01), PAG (MEM-255), CD3 (MEM-57), CD5 (MEM-32), Lck (LCK-01), ZAP-70 (ZAP-03), CD69 (FN50, Alexa Fluor 647), tubulin (TU-01), and LAMP-1 (CD107a, B-T47) from Exbio (Vestec, Czech Republic); GAPDH from Sigma; Src (N-16), c-Jun (N), ERK2 (C-14), TCRζ (6B10.2) from Santa Cruz Biotechnology (Santa Cruz, CA); IL-2 (MQ1–17H12, APC) from eBioscience (San Diego, CA); phosphotyrosine (4G10) from Upstate Biotechnology (Lake Placid, NY); HA tag (6E2), pZAP-70 Tyr-319, pPLCγ1 Tyr-783, PLCγ1, pp42/44 ERK1/2 (Thr-202/Tyr-204), pp38 MAPK (T180/Y182), p38 MAPK, pJNK (T183/Y185), JNK, pc-Jun (Ser-63/Ser-73), pSFK (Y416), and pLck (Y505) from Cell Signaling Technology (Beverly, MA). The following inhibitors were used: the inhibitor of the Src family kinases, PP2 (Calbiochem), final concentration 10 μm and the pan-caspase inhibitor, Z-VAD-FMK (Alexis Biochemicals), final concentration 10 μm. Z-VAD-FMK inhibitor assay was performed using 2.5 × 105 cells in 96-well tissue culture plates in triplicates. Viability was assessed by flow cytometry (propidium iodide exclusion and side plus forward scatter gating). Jurkat cells (107) were transfected with 15 μg of the corresponding plasmid DNA in 300 μl of RPMI medium supplemented with 10% FCS by electroporation (250 V, 950 microfarads) using a GenePulser electroporator (Bio-Rad). For the pZRD-inducible system (21Lessard J. Aicha S.B. Fournier A. Calvo E. Lavergne E. Pelletier M. Labrie C. Prostate. 2007; 67: 808-819Crossref PubMed Scopus (13) Google Scholar), based on the RheoSwitch Mammalian Inducible Expression System (New England Biolabs, Ipswich, MA), transfectants were selected in the culture medium containing Zeocin (200 μg/ml, Invitrogen). Stably transfected GFP-positive cells were sorted and further subcloned by limiting dilution. The clones (denoted J-iPRR7) that expressed PRR7 only after induction with RSL1 (500 nm for maximal induction, RheoSwitch Ligand 1, New England Biolabs) were used in further experiments. COS-7 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. To analyze PRR7 up-regulation, freshly isolated PBL were stimulated using 1) anti-CD3 IgG (MEM-57) immobilized on tissue culture plastic (10 μg/ml) and soluble anti-CD28 IgM (100× diluted hybridoma supernatant), 2) PHA-L (2.5 μg/ml, Sigma), and 3) a combination of PMA (10 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma). For short term activation (Ca2+ flux, phospho-Tyr blots), Jurkat cells were stimulated with anti-TCR IgM (C305) (23Fraser J.D. Goldsmith M.A. Weiss A. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 7133-7137Crossref PubMed Scopus (6) Google Scholar) (10 μg/ml) at 37 °C for 2 min. For activation of Jurkat cells in an IL-2 production assay, the combination of PMA and ionomycin was used as described above. Generally, 5 × 107 cells were lysed in 1 ml of lysis buffer (20 mm Tris pH 7.5, 100 mm NaCl, 5 mm iodoacetamide, 10 mm EDTA, 50 mm NaF, 10 mm Na4P2O7, 10% v/v glycerol, and Protease Inhibitor Mixture III, Calbiochem) containing 1% w/v detergent LM (Calbiochem) or Brij-98 (Sigma) for 30 min on ice. To remove nuclei and other insoluble materials, the lysate was spun down at 16,000 × g for 10 min at 4 °C. Density gradient ultracentrifugation and gel filtration on Sepharose 4B were performed as previously described (24Otáhal P. Angelisová P. Hrdinka M. Brdicka T. Novák P. Drbal K. Horejsí V. J. Immunol. 2010; 184: 3689-3696Crossref PubMed Scopus (32) Google Scholar). For immunoprecipitation experiments, we coupled the anti-PRR7 IgG (TRAP3-03) to CNBr-Sepharose 4B (Amersham Biosciences) or used soluble antibodies and Protein A/G PLUS-agarose IP Reagent (Santa Cruz). Palmitoylation of PRR7 was examined using the acyl-biotinyl exchange chemistry-based method (25Wan J. Roth A.F. Bailey A.O. Davis N.G. Nat. Protoc. 2007; 2: 1573-1584Crossref PubMed Scopus (304) Google Scholar). Briefly, plasma membranes from 5 × 107 cells were isolated, and palmitate protein modifications were removed by hydroxylamine and replaced with biotin. Biotinylated proteins were then immunoprecipitated on streptavidin-agarose beads. Other biochemical methods (SDS-PAGE, immunoblotting) were performed essentially as described before (4Brdicka T. Pavlistová D. Leo A. Bruyns E. Korínek V. Angelisová P. Scherer J. Shevchenko A. Hilgert I. Cerný J. Drbal K. Kuramitsu Y. Kornacker B. Horejsí V. Schraven B. J. Exp. Med. 2000; 191: 1591-1604Crossref PubMed Scopus (405) Google Scholar). Jurkat cells were allowed to adhere to polylysine-coated coverslips for 30 min, then fixed with 4% w/v formaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 (Sigma) for 5 min. Blocking was performed in 2.5% BSA and 10% goat serum (Sigma) in PBS for 30 min. Cells were then incubated with 100×-diluted LAMP-1 antibody followed by 750×-diluted Alexa 647-labeled goat anti-mouse IgG secondary antibody (Molecular Probes, Invitrogen). The DNA dye, Hoechst 33258 (1 μg/ml, Invitrogen), was used to visualize nuclei. Images were captured with a Leica SP5 confocal microscope and a 63× objective lens (Leica Microsystems, Mannheim, Germany). CD69 surface staining and intracellular IL-2 staining were done according to the standard protocols. To measure apoptosis in cells expressing PRR7, we performed annexin V/Dy-647 (Apronex Biotechnologies, Vestec, Czech Republic) staining in combination with the DNA dye, Hoechst 33258, according to the manufacturer's instructions. To measure calcium response to anti-TCR activation, cells were first loaded with 5 μm Fura Red (Molecular Probes, Invitrogen) in loading buffer (1 × Hanks' balanced salt solution, 2% FCS, without Ca2+ or Mg2+) for 30 min at 37 °C. After washing, cells were resuspended in loading buffer supplemented with Ca2+ and Mg2+ and kept on ice. Cells were warmed up for 10 min at 37 °C and then stimulated with an irrelevant control antibody or the anti-TCR C305 mAb (10 μg/ml). Calcium flux was monitored for 240 s. Flow cytometry was carried out on an LSRII instrument (BD Biosciences), and cells were sorted on FACSVantage (BD Biosciences). Analysis of the data was performed using FlowJo software (Tree Star, Ashland, OR) as specified in the figure legends (FIGURE 6, FIGURE 7, FIGURE 8).FIGURE 7Tyrosine phosphorylation of PRR7 and association of PRR7 with Src. A, J-iPRR7 cells were induced to express PRR7 and simultaneously treated with the SFK inhibitor, PP2 (10 μm), for 18 h or left untreated. Tyrosine phosphorylation status of PRR7 was analyzed by immunoblotting with the anti-phospho-Tyr mAb (4G10). GAPDH staining served as a sample loading control. B, PRR7 was co-transfected with Src kinases into COS-7 cells. After 24 h, cells were lysed, and tyrosine phosphorylation of PRR7 was detected by immunoblotting with the anti-phospho-Tyr (4G10) mAb. C, J-iPRR7 cells were induced to express PRR7 or left uninduced for 18 h and lysed in LM buffer, and PRR7 was immunoprecipitated (IP) using the anti-PRR7-Sepharose or an isotype control mAb. Eluted proteins were analyzed by immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Activated phenotype and impaired TCR signaling in J-iPRR7 cells. A, J-iPRR7 cells were induced for 12 h to express PRR7, stained for the surface lymphocyte activation marker, CD69, and analyzed by flow cytometry. B, J-iPRR7 cells were induced to express PRR7. After 12 h, the cells were stimulated for additional 6 h with PMA and ionomycin in the presence of brefeldin A (5 μg/ml), fixed, stained for cytokine IL-2, and analyzed by flow cytometry. C, J-iPRR7 cells were induced for 16 h to express PRR7 and then loaded with a calcium indicator dye Fura Red. Calcium response after the anti-TCR IgM (C305, 10 μg/ml) stimulation was measured by flow cytometry. Collected data were analyzed in FlowJo software and plotted as mean fluorescence values. D, J-iPRR7 cells were induced to express PRR7 (16 h). Cells were stimulated with the anti-TCR IgM (C305, 10 μg/ml, 2 min) or left unstimulated. Whole cell lysates were analyzed by immunoblotting with the anti-phospho-Tyr mAb (4G10). Membrane was reprobed with GAPDH antibody as a loading control. E, J-iPRR7 cells were treated as in panel D, and lysate was subjected to immunoprecipitation (IP) with the anti-PRR7 mAb. Immunoprecipitated material was analyzed by immunoblotting with the anti-phospho-Tyr mAb (4G10) and the anti-PRR7 mAb.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Human PRR7 (18Murata Y. Doi T. Taniguchi H. Fujiyoshi Y. Biochem. Biophys. Res. Commun. 2005; 327: 183-191Crossref PubMed Scopus (22) Google Scholar) is a 274-aa protein with a sequence typical for TRAPs. It has a short N-terminal extracellular peptide, a single transmembrane segment, and a cytoplasmic part containing several conserved binding motifs (Fig. 1A). These motifs include multiple SH2 domain binding and/or endocytic tyrosine-based motifs (YXX(I/L/V/A)), multiple proline-rich SH3 binding motifs (PXXP), group I WW domain binding motifs (PPXY), and a C-terminal class I PDZ domain binding motif (TTAV). In addition, PRR7 contains a potential submembrane palmitoylation motif (CCXC, Fig. 1A). PRR7 is a highly conserved protein. The percentage of aa identity is more than 94% among available sequences from placental mammals, and a substantial level of aa identity is observed even if lower vertebrate species (amphibian, fish) are included (Table 1). The highest degree of conservation of PRR7 protein sequence was observed in the extracellular, transmembrane, and submembrane parts, including the palmitoylation motif. In addition, the region containing three tyrosine residues, Tyr-153, Tyr-166, and Tyr-177, and the C terminus with the PDZ domain binding motif also displayed a very high degree of homology (Fig. 1B). Interestingly, N-terminal part of PRR7 as well as sequences surrounding tyrosines 153 and 166 form a putative domain identified in Pfam data base (26Finn R.D. Mistry J. Tate J. Coggill P. Heger A. Pollington J.E. Gavin O.L. Gunasekaran P. Ceric G. Forslund K. Holm L. Sonnhammer E.L. Eddy S.R. Bateman A. Nucleic Acids Res. 2010; 38: D211-D222Crossref PubMed Scopus (2484) Google Scholar) as the WBP-1 domain (PF11669). Among other proteins, this domain is present in WW domain-binding protein 1 (WBP-1) where it mediates the interaction with WW domains of Yes-associated protein (YAP) via tandem PPXY motifs in its C terminus (27Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 7819-7823Crossref PubMed Scopus (489) Google Scholar). Similar motifs encompass tyrosines 153 and 166 in PRR7, suggesting possible involvement of this region in an interaction with a WW domain containing protein (Fig. 1C).TABLE 1Conservation of PRR7 protein in vertebratesSpeciesLengthIdentityaa%Pongo pygmaeus (orangutan)27399Pan troglodytes (chimpanzee)25998Macaca mulatta (macaque)27698Rattus norvegicus (rat)26998M. musculus (mouse)26997Spermophilus tridecemlineatus (squirrel)26997Canis familiaris (dog)27297Bos taurus (cattle)26997Echinops telfairi (tenrec)27095Sus scrofa (pig)26994Monodelphis domestica (opossum)28081X. tropicalis (frog)22756D. rerio (fish)24648Gasterosteus aculeatus (fish)27944 Open table in a new tab To determine the PRR7 expression pattern in human tissues, we performed RT-qPCR on a panel of multiple tissue RNAs. As shown in Fig. 2A, PRR7 mRNA is expressed most strongly in brain tissue and moderately in several other tissues: esophagus, trachea, lung, ovary, cervix, prostate, testes, thyroid, including the immune organs thymus and lymph nodes. We also found low levels of PRR7 mRNA in a number of human cell lines (Jurkat, U937, THP-1, MEG-01, CaCo2, HEK293FT); the only cell line exhibiting relatively high expression was T cell line MOLT-4 (Fig. 2B). We next analyzed whether the expression level of PRR7 mRNA in PBL changes after activation. Interestingly, we observed rapid up-regulation of PRR7 mRNA in PHA-stimulated PBL. It was detectable at 1 h and peaked at 8 h after stimulation (Fig. 2C). To examine the expression of PRR7 at the protein level, we generated anti-human PRR7 monoclonal antibodies. Unfortunately, none of the generated antibodies could clearly detect PRR7 after immunoblotting of whole primary cell lysates (except for mouse or rat brain tissues; not shown). Moreover, native PRR7 could not be quantitatively immunoprecipitated from cell lysates using these antibodies, suggesting partial blocking of the epitope (not shown). Nevertheless, it was still possible to use immunoprecipitation to concentrate PRR7 protein to the degree sufficient for detection by immunoblotting also in leukocytes. We then used this approach to verify our results from previous experiments. As shown in Fig. 2D, PRR7 protein was detected in PBL activated by various stimuli, including activation with anti-CD3 and anti-CD28 antibodies, PHA, or PMA and ionomycin. We could also detect PRR7 protein in several lymphoid cell lines, including Jurkat, Ramos, and MOLT-4 (Fig. 2E). The highest protein levels were observed in MOLT-4, which correlated well with the higher PRR7 mRNA level in this cell line (Fig. 2, B and E). Because the level of PRR7 expression in leukocytes (cell lines and primary PBL) was low and its direct immunoprecipitation was inefficient, we generated multiple Jurkat cell lines transfected with PRR7 constructs containing a C-terminal GFP tag. Surprisingly, we were not able to establish a Jurkat cell line with a sufficiently high expression of PRR7. Direct observation of the PRR7-GFP-transfected Jurkat cells excluded a rapid necrotic cell death of the transfectants. Instead, we consistently observed a gradual loss of the PRR7 transfectants from the culture within a few days (not shown). PRR7high cells were stained with the DNA dye, propidium iodide, and analysis of the staining suggested that overexpression of PRR7 protein caused apoptosis (not shown). To exclude the possibility that the apoptosis induction is at least in part the result of the stress caused by the transfection procedure (electroporation), we generated a stable Jurkat clone (J-iPRR7) inducibly expressing full-length HA-tagged PRR7. In this case a modified RheoSwitch Mammalian Inducible Expression System (21Lessard J. Aicha S.B. Fournier A. Calvo E. Lavergne E. Pelletier M. Labrie C. Prostate. 2007; 67: 808-819Crossref PubMed Scopus (13) Google Scholar) was utilized. It allowed us to tightly regulate the expression of PRR7 using the synthetic compound RSL1. Without RSL1, PRR7 expression was not detectable; maximal expression was achieved in the presence of RSL1 12 h after induction (Fig. 3A). Strikingly, we observed that in J-iPRR7 cells, apoptosis could be clearly detected within 24 h of induction by RSL1 and gradually increased with time (Fig. 3, B and C), suggesting that apoptosis induction was a direct consequence of PRR7 expression. This process could be to a substantial degree inhibited by caspase inhibitor Z-VAD, demonstrating the involvement of caspase-dependent apoptotic pathways (Fig. 3D). To identify the regions of the PRR7 molecule involved in the apoptosis induction, we generated a set of Jurkat cell lines transiently expressing either full-length PRR7-GFP or its mutants gradually truncated from the C terminus (Fig. 3E). We also generated a mutant where all the tyrosines in the PRR7 sequence were mutated to phenylalanines (all Y to F) and a mutant lacking the N terminus, including extracellular peptide, transmembrane segment, a" @default.
• W2079200708 created "2016-06-24" @default.
• W2079200708 creator A5017454714 @default.
• W2079200708 creator A5046384096 @default.
• W2079200708 creator A5046576229 @default.
• W2079200708 creator A5048076007 @default.
• W2079200708 creator A5049990120 @default.
• W2079200708 creator A5058652447 @default.
• W2079200708 creator A5064911651 @default.
• W2079200708 creator A5071573004 @default.
• W2079200708 creator A5078265203 @default.
• W2079200708 creator A5088258486 @default.
• W2079200708 date "2011-06-01" @default.
• W2079200708 modified "2023-10-17" @default.
• W2079200708 title "PRR7 Is a Transmembrane Adaptor Protein Expressed in Activated T Cells Involved in Regulation of T Cell Receptor Signaling and Apoptosis" @default.
• W2079200708 cites W1493638086 @default.
• W2079200708 cites W1521372932 @default.
• W2079200708 cites W1534253341 @default.
• W2079200708 cites W1548078808 @default.
• W2079200708 cites W1576055832 @default.
• W2079200708 cites W1636129643 @default.
• W2079200708 cites W1845579190 @default.
• W2079200708 cites W1974681499 @default.
• W2079200708 cites W1978222385 @default.
• W2079200708 cites W1980129122 @default.
• W2079200708 cites W1988319136 @default.
• W2079200708 cites W1992970966 @default.
• W2079200708 cites W1994745798 @default.
• W2079200708 cites W2002573954 @default.
• W2079200708 cites W2014177218 @default.
• W2079200708 cites W2033152875 @default.
• W2079200708 cites W2039998576 @default.
• W2079200708 cites W2051052911 @default.
• W2079200708 cites W2051972593 @default.
• W2079200708 cites W2054458458 @default.
• W2079200708 cites W2065007544 @default.
• W2079200708 cites W2084186633 @default.
• W2079200708 cites W2103967533 @default.
• W2079200708 cites W2106198450 @default.
• W2079200708 cites W2113809180 @default.
• W2079200708 cites W2116862638 @default.
• W2079200708 cites W2129108588 @default.
• W2079200708 cites W2138773717 @default.
• W2079200708 cites W2147679202 @default.
• W2079200708 cites W2148351021 @default.
• W2079200708 cites W2148670260 @default.
• W2079200708 cites W2152770371 @default.
• W2079200708 cites W2160675888 @default.
• W2079200708 cites W2170848162 @default.
• W2079200708 cites W2171480972 @default.
• W2079200708 cites W4210623056 @default.
• W2079200708 cites W95054676 @default.
• W2079200708 doi "https://doi.org/10.1074/jbc.m110.175117" @default.
• W2079200708 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3103341" @default.
• W2079200708 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21460222" @default.
• W2079200708 hasPublicationYear "2011" @default.
• W2079200708 type Work @default.
• W2079200708 sameAs 2079200708 @default.
• W2079200708 citedByCount "12" @default.
• W2079200708 countsByYear W20792007082012 @default.
• W2079200708 countsByYear W20792007082014 @default.
• W2079200708 countsByYear W20792007082016 @default.
• W2079200708 countsByYear W20792007082017 @default.
• W2079200708 countsByYear W20792007082018 @default.
• W2079200708 countsByYear W20792007082021 @default.
• W2079200708 countsByYear W20792007082022 @default.
• W2079200708 crossrefType "journal-article" @default.
• W2079200708 hasAuthorship W2079200708A5017454714 @default.
• W2079200708 hasAuthorship W2079200708A5046384096 @default.
• W2079200708 hasAuthorship W2079200708A5046576229 @default.
• W2079200708 hasAuthorship W2079200708A5048076007 @default.
• W2079200708 hasAuthorship W2079200708A5049990120 @default.
• W2079200708 hasAuthorship W2079200708A5058652447 @default.
• W2079200708 hasAuthorship W2079200708A5064911651 @default.
• W2079200708 hasAuthorship W2079200708A5071573004 @default.
• W2079200708 hasAuthorship W2079200708A5078265203 @default.
• W2079200708 hasAuthorship W2079200708A5088258486 @default.
• W2079200708 hasBestOaLocation W20792007081 @default.
• W2079200708 hasConcept C170493617 @default.
• W2079200708 hasConcept C183786373 @default.
• W2079200708 hasConcept C185592680 @default.
• W2079200708 hasConcept C190283241 @default.
• W2079200708 hasConcept C24530287 @default.
• W2079200708 hasConcept C55493867 @default.
• W2079200708 hasConcept C62478195 @default.
• W2079200708 hasConcept C86803240 @default.
• W2079200708 hasConcept C95444343 @default.
• W2079200708 hasConceptScore W2079200708C170493617 @default.
• W2079200708 hasConceptScore W2079200708C183786373 @default.
• W2079200708 hasConceptScore W2079200708C185592680 @default.
• W2079200708 hasConceptScore W2079200708C190283241 @default.
• W2079200708 hasConceptScore W2079200708C24530287 @default.
• W2079200708 hasConceptScore W2079200708C55493867 @default.
• W2079200708 hasConceptScore W2079200708C62478195 @default.
• W2079200708 hasConceptScore W2079200708C86803240 @default.
• W2079200708 hasConceptScore W2079200708C95444343 @default.
• W2079200708 hasIssue "22" @default.
• W2079200708 hasLocation W20792007081 @default.

• next