Evidence of Functional Modulation of the MEKK/JNK/cJun Signaling Cascade by the Low Density Lipoprotein Receptor-related Protein (LRP)
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m204426200
ISSN1083-351X
AutoresChristina Maria Lutz, Johannes Nimpf, Marcel Jenny, Karl Boecklinger, Christiane Enzinger, Gerd Utermann, Gabriele Baier‐Bitterlich, Gottfried Baier,
Tópico(s)Melanoma and MAPK Pathways
ResumoLipoprotein receptors, such as LRP, have been shown to assemble multiprotein complexes containing intracellular signaling molecules; however, in vivo, their signaling function is poorly understood. Using a novel LRP receptor fusion construct, a type I transmembrane protein chimera, termed sIgG-LRP (bearing the intracellular COOH-terminal tail of human LRP as recombinant fusion to a transmembrane region plus the extracellular IgG-Fc domain), we here investigated LRP signal transduction specificity in intact cells. First and similar to activated α2-macroglobulin as agonist of endogenous LRP, expression of sIgG-LRP demonstrated significant apoptosis protection. Second and similar to α2-macroglobulin-induced endogenous LRP, sIgG-LRP is sufficient to negatively modulate mitogen-induced Elk-1 and cJun (but not NF-κB) transcriptional activity. Third, expression of sIgG-LRP also impaired cJun transactivation mediated by constitutive active mutants of Rac-1 and MEKK-1. Fourth and unexpectedly, sIgG-LRP expression was found to be associated with a marked enhancement of mitogen-induced cJun amino-terminal kinase (JNK) activation. Fifth, confocal microscopic examination and subcellular fractionation demonstrated that sIgG-LRP and JNK co-localize in transfected cells. Therefore, sIgG-LRP expression was found to significantly impair activation-induced translocation of JNK into the nucleus. Taken together, we here demonstrate that sIgG-LRP protein sequesters activated JNK into the plasma membrane compartment in intact cells, inhibiting nuclear activation of the JNK-dependent transcription factors Elk-1 and cJun. Lipoprotein receptors, such as LRP, have been shown to assemble multiprotein complexes containing intracellular signaling molecules; however, in vivo, their signaling function is poorly understood. Using a novel LRP receptor fusion construct, a type I transmembrane protein chimera, termed sIgG-LRP (bearing the intracellular COOH-terminal tail of human LRP as recombinant fusion to a transmembrane region plus the extracellular IgG-Fc domain), we here investigated LRP signal transduction specificity in intact cells. First and similar to activated α2-macroglobulin as agonist of endogenous LRP, expression of sIgG-LRP demonstrated significant apoptosis protection. Second and similar to α2-macroglobulin-induced endogenous LRP, sIgG-LRP is sufficient to negatively modulate mitogen-induced Elk-1 and cJun (but not NF-κB) transcriptional activity. Third, expression of sIgG-LRP also impaired cJun transactivation mediated by constitutive active mutants of Rac-1 and MEKK-1. Fourth and unexpectedly, sIgG-LRP expression was found to be associated with a marked enhancement of mitogen-induced cJun amino-terminal kinase (JNK) activation. Fifth, confocal microscopic examination and subcellular fractionation demonstrated that sIgG-LRP and JNK co-localize in transfected cells. Therefore, sIgG-LRP expression was found to significantly impair activation-induced translocation of JNK into the nucleus. Taken together, we here demonstrate that sIgG-LRP protein sequesters activated JNK into the plasma membrane compartment in intact cells, inhibiting nuclear activation of the JNK-dependent transcription factors Elk-1 and cJun. low density lipoprotein receptor-related protein low-density lipoprotein amyloid precursor protein Alzheimer's disease constitutive active extracellular signal regulated kinase cJun NH2-terminal kinase JNK interacting protein mitogen-activated protein kinase nuclear factor of κB phorbol 12-myristate 13-acetate: MEKK, MEK kinase nerve growth factor phosphate-buffered saline fluorescence-activated cell sorter 4-morpholinepropanesulfonic acid hemagglutinin phosphatidylinositol 3-kinase Low density lipoprotein receptor-related protein (LRP)1 is one member of the LDL receptor family that also includes the LDL receptor, the very low density lipoprotein receptor, megalin, LRP5, LRP6, and apoER2 receptor (see Ref. 1Herz J. Neuron. 2001; 29: 571-581Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar for review). These multifunctional lipoprotein receptors are established cargo transporters, but expression level at the cell surface and/or (ant)agonistic binding of diverse biological ligands are now thought to potentially evoke signaling pathways involved in cell fate determination (see Ref. 2Herz J. Beffert U. Nat. Rev. Neurosci. 2000; 1: 51-58Crossref PubMed Scopus (171) Google Scholar for review). LRP is expressed abundantly in neurons and microglia of the central nervous system (3Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1055) Google Scholar, 4Marzolo M.P. von Bernhardi R., Bu, G. Inestrosa N.C. J. Neurosci. Res. 2000; 60: 401-411Crossref PubMed Scopus (79) Google Scholar). Disruption of the LRP gene in mice blocks development of LRP−/− embryos around the implantation (5Herz J. Clouthier D.E. Hammer R.E. Cell. 1992; 71: 411-421Abstract Full Text PDF PubMed Scopus (501) Google Scholar). However, the complex phenotype of the few malformed LRP-deficient embryos that survive until E10 (6May P. Reddy Y.K. Herz J. J. Biol. Chem. 2002; 277: 18736-18743Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), similar to the very low density lipoprotein ApoER2 receptor double knockout phenotype (7Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar), postulated some LRP receptor signaling function(s). Consistently, LRP and several of its ligands, including α2-macroglobulin, tissue plasminogen activator (tPA), apoE-containing lipoproteins, and the amyloid precursor protein (APP) (8Willnow T.E. Orth K. Herz J. J. Biol. Chem. 1994; 269: 15827-15832Abstract Full Text PDF PubMed Google Scholar, 9Kounnas M.Z. Moir R.D. Rebeck G.W. Bush A.I. Argraves W.S. Tanzi R.E. Hyman B.T. Strickland D.K. 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Ranganathan S. Kuznetsov S. Gorlatov N. Migliorini M.M. Loukinov D. Ulery P.G. Mikhailenko I. Lawrence D.A. Strickland D.K. J. Biol. Chem. 2002; 277: 15499-15506Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), the non receptor tyrosine kinases Src and Fyn (14Boucher P. Liu P. Gotthardt M. Hiesberger T. Anderson R.G. Herz J. J. Biol. Chem. 2002; 277: 15507-15513Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), and the JNK-interacting proteins (JIP-1 & 2) (15Gotthardt M. Trommsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 16Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), which act as molecular scaffolds for the JNK signaling pathway (see Ref. 17Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4307) Google Scholar for review). Quite similarly, such intracellular signaling protein complexes have been found for APP (7Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 11Kinoshita A. Whelan C.M. Smith C.J. Mikhailenko I. Rebeck G.W. Strickland D.K. Hyman B.T. J. Neurosci. 2001; 21: 8354-8361Crossref PubMed Google Scholar, 18Russo T. Faraonio R. Minopoli G., De Candia P., De Renzis S. Zambrano N. FEBS Lett. 1998; 434: 1-7Crossref PubMed Scopus (97) Google Scholar, 19Matsuda S. Yasukawa T. Homma Y. Ito Y. Niikura T. Hiraki T. Hirai S. Ohno S. Kita Y. Kawasumi M. Kouyama K. Yamamoto T. Kyriakis J.M. Nishimoto I. J. Neurosci. 2001; 21: 6597-6607Crossref PubMed Google Scholar, 20Scheinfeld M.H. Roncarati R. Vito P. Lopez P.A. Abdallah M. D'Adamio L. J. Biol. Chem. 2002; 277: 3767-3775Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and presumably for its closely related homologues amyloid precursor-like proteins 1 and 2. Nevertheless, the cellular signaling function of these receptor families are not well understood, and no systematic investigation on downstream effector functions has yet been performed. Studies presented here are focused on the elucidation of intracellular key signaling pathways that occur downstream of LRP (as one representative member of this family) and in direct comparison downstream of APP activation. Given the complex situation of the LRP ectodomain binding protein network, we here employed the established chimeric receptor approach (21Romeo C. Kolanus W. Amiot M. Seed B. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 117-125Crossref PubMed Google Scholar, 22Kolanus W. Romeo C. Seed B. Cell. 1993; 74: 171-183Abstract Full Text PDF PubMed Scopus (301) Google Scholar), fusing a transmembrane region and the extracellular sIgG Fc-domain to the intracellular tails of APP and LRP, respectively. Our results indicate that in the transfected Jurkat TAg cell line ectopic expression of the cytoplasmic domain of LRP (but not or at least much less of APP), as a specific constituent downstream or parallel of MEKK-1, exerts modifying signaling functions and that active JNK is sequestered by plasma membrane resident LRP multiprotein complexes, preventing JNK from translocation into and transactivation of Elk-1 and cJun within the nucleus. Thus, this study formally demonstrates, for the first time, that lipoprotein receptors (via the established interaction with JIP) (15Gotthardt M. Trommsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 16Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) modulate mitogenic signaling function(s), e.g.scaffold JNK in intact cells, providing a novel insight into the role of JNK sequestration and subsequent inhibition of nuclear JNK target substrates, cJun- and Elk-1 transactivation by LRP. Camptothecin was purchased from Molecular Probes Inc. (Eugene, OR). Phorbol-12-myristate 13-acetate (PMA), ionomycin, NGF, and native α2-macroglobulin were purchased from Sigma. Synthetic NF-κB-Luc construct and the Elk-1 and c-Jun PathDetectTM transreporting systems have been obtained from Stratagene (La Jolla, CA). The plasmid encoding the murine major histocompatibility complex class I protein H2-Kk was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The plasmids pEF-RacVI2, MEKK-1 catalytic fragment (Δ), JNK-2-HA, ERK-2-MYC, and pEF-bcl-2 were described recently (23Ghaffari-Tabrizi N. Bauer B. Villunger A. Baier-Bitterlich G. Altman A. Utermann G. Uberall F. Baier G. Eur. J. Immunol. 1999; 29: 132-142Crossref PubMed Scopus (108) Google Scholar,24Villunger A. Ghaffari-Tabrizi N. Tinhofer I. Krumbock N. Bauer B. Schneider T. Kasibhatla S. Greil R. Baier-Bitterlich G. Uberall F. Green D.R. Baier G. Eur. J. Immunol. 1999; 29: 3549-3561Crossref PubMed Scopus (47) Google Scholar). The plasmid pJIP-1 was a kind gift from Gerard Waeber, Lausanne, Switzerland (25Bonny C. Nicod P. Waeber G. J. Biol. Chem. 1998; 273: 1843-1846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). For activation, α2-macroglobulin was incubated in 2m methylamine/50 mm Tris-HCl (pH 8.0)/150 mm NaCl for 2 h at room temperature in the dark. Unreacted methylamine was removed by dialysis for 48 h with 5 changes of HEPES buffer (20 mm HEPES/NaOH, pH 7.4, 150 mm NaCl) at 4 °C. Finally, α2-macroglobulin preparation was dialyzed again with serum-free BME Eagle W medium for 4 h and sterilized through a 0.22-μm filter and then stored at 4 °C for up to 2 weeks. Using recombinant PCR, appropriate restriction sites have been introduced into the human APP and LRP cDNA sequences (employing primers LRP-5′-MluI: 5′-GTATTCACGCGTAAGCGGCGAGTCCAAGGGGC-3′; LRP-3′-NotI: 5′-TCCGACGCGGCCGCCTACTATGCCAAGGGGTCCCCTATC-3′, and APP-5′-MluI: 5′-TTGGTGACGCGTAAGAAGAAACAGTACACATCCATTC-3′, APP-3′-NotI: 5′-GCTGCTGCGGCCGCCTACTAGTTCTGCATCTGCTCAAAGAA-3′ respectively). PCR fragments were purified and directionally ligated in frame into theMluI/NotI-linearized and phosphatase-treated p5C7 vector encoding the leader sequence from human CD5, the CD7 transmembrane region, and the extracellular CH2 and CH3 heavy chain domains from human IgG1 (kind gift by Dr. Kolanus, Munich, Germany) (26Zeitlmann L. Knorr T. Knoll M. Romeo C. Sirim P. Kolanus W. J. Biol. Chem. 1998; 273: 15445-15452Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), generating sIgG-receptor fusion mutant expression constructs. Correct constructs have been identified and confirmed by restriction analysis and partial DNA sequencing employing primers p5C7-sense: 5′-TGCCCTCCCTGCGGCCCT-3′ and p5C7-antisense: 5′-TTACCAATGATTATTTCGTT-3′. For reporter gene analysis, anti-Fc antibody-mediated cross-linking was done for 16 h employing Dynabeads-Protein A (Dynal, Hamburg Germany) coated with rabbit anti-human IgG. For apoptosis detection soluble goat anti-human IgG was used for cross-linking. Cerebellar neurons were prepared from the cerebelli of 5–7-day-old beheaded rats (as described by Ref. 27Hatten M.E. J. Cell Biol. 1985; 100: 384-396Crossref PubMed Scopus (501) Google Scholar) and cultivated in BME medium (complemented with 10% heat-inactivated fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 30 mm glucose, and 20 mm KCl) on poly-l-lysine-coated 6-well plates at a density of 1 × 106/well at 37 °C and 5% CO2. PC12 cells were cultivated in RPMI 1640 medium complemented with 2 mm glutamine, 100 units/ml penicillin, 5% fetal calf serum, 0.1 mg/ml streptomycin, and 10% heat-inactivated horse serum. Cells were fed every second day and split once a week (split ratio 1:10). Culture plates had to be coated with collagen S (120 μg/ml in distilled water). 24 h after medium exchange cells were harvested and washed in prewarmed PBS (10 min, 200 g, room temperature). 2 × 106 cells (resuspended in RPMI medium without supplements) were mixed with up to 45 μg of plasmid DNA (total volume 400 μl) and transferred into cuvettes and incubated at room temperature for 15 min. The electroporation was performed in an Electro Cell Manipulator 600 using predetermined optimal conditions (250V, 48Ω, 1100 microfarads) (28Enzinger C. Wirleitner B. Lutz C. Bock G. Tomaselli B. Baier G. Fuchs D. Baier-Bitterlich G. Eur J. Cell Biol. 2002; 81: 97-202Crossref Scopus (6) Google Scholar). After transfection cells were incubated at room temperature for an additional 15 min before cultured in RPMI medium with supplements at 37 °C. CD4+ Jurkat TAg T cells (a kind gift from Dr. G. Crabtree, Stanford University, Stanford, CA) that stably express the SV40-derived large T antigen were cultivated in RPMI 1640 medium complemented with 2 mm glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal calf serum. 24 h after medium change Jurkat TAg cells were harvested by centrifugation, washed three times in medium without supplements, resuspended at a concentration of 2 × 107 cells/400 μl, and incubated on ice for 15 min. Transient transfection of cells was performed by electroporation in a BTX T820 ElectroSquarePorator (ITC, Biotech, Heidelberg, Germany) apparatus using predetermined optimal conditions (volume, 400 μl of cell suspension; mode, 99 μs; voltage, 450 V; number of pulses, 5). After electroporation cells were incubated for 15 min at room temperature before cultured in RPMI medium with supplements at 37 °C. Reporter gene expression was measured in co-transfection assays using 20–30 μg of the sIgG expression constructs and 10 μg of NF-κB-Luc or 16 μg of pFRLuc promoter firefly luciferase reporter (RLU1) plus 4 μg of pFA-Jun or pFA Elk-1, respectively, as described in Ref. 29Bauer B. Krumbock N. Ghaffari-Tabrizi N. Kampfer S. Villunger A. Wilda M. Hameister H. Utermann G. Leitges M. Uberall F. Baier G. Eur. J. Immunol. 2000; 30: 3645-3654Crossref PubMed Scopus (48) Google Scholar. After 30 h cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for up to 16 h or left unstimulated (Me2SO buffer control), as indicated. To allow a comparison between different experiments, which vary with regard to transfection efficiencies, the different cell populations to be tested were co-transfected with a housekeeping expression plasmid (0.3 μg of the Renilla luciferase reporter vector pTK-Renilla-Luc, RLU2, Promega, Madison, WI) driven by the Herpes simplex virus thymidine kinase promoter, and results of enhancer/promoter activation (i.e. firefly luciferase reporter protein expression) were normalized to the corresponding Renilla luciferase reporter. Luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) and a 1450 Micro-Beta WALLAC Jet Liquid Scintillation and Luminescence Counter. PC12 cells were stimulated in triplicates with 5 μg/ml NGF-β for 16 h for reporter gene analysis. 50 μg/ml activated or native α2-macroglobulin were added. The results shown are obtained with different preparations of expression plasmids and represent the mean ± S.E. from representative experiments done in duplicates. sIgG surface expression of transfected cells was detected with goat anti-human IgG (Dianova, Hamburg, Germany) and, as secondary reagent, donkey anti-goat IgG-fluorescein isothiocyanate (Jackson ImmunoResearch), each employed for 30 min at 4 °C and analyzed by FACS scan. SDS-PAGE was performed under reducing conditions using 4–12% Bis/Tris-buffered gels (Novex, San Diego, CA) and MOPS-running buffer for 90 min at 150 V. Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA) by semi-dry blotting (90 mA/112 cm2, 90 min, room temperature). Blots were blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% non-fat dry milk for 1 h at room temperature, incubated with primary antibodies over night at 4 °C and peroxidase-conjugated secondary antibodies (Pierce, 1:5.000) for 1 h at room temperature. For antigen detection enhanced chemiluminescence was used (Super Signal, Pierce). Cerebellar neurons were cultivated 4 days in full BME medium (on poly-l-lysine-coated glass cover slips) and then preincubated with 50 μg/ml activated or inactivated α2-macroglobulin for 16 h. Cells were washed once with prewarmed PBS and incubated with supplemented medium containing 8 μm Hoechst 33342 for 15 min at 37 °C in the dark. 5 μg/ml propidium iodide were added, and cells incubated for an additional 5 min. Cells were washed twice in PBS and counted in an axioplan fluorescence microscope (PI: excitation: 570 nm, emission: 590 nm/Hoechst: excitation: 400 nm, emission: 420 nm) and pictures were taken with an Olympus U-CMAD-2 camera. Jurkat TAg cells were co-transfected with 20 μg of the indicated pEF-neomycin empty vector control, bcl-2, or sIgG expression constructs and the surface marker expression plasmid encoding the murine major histocompatibility complex class I H2-Kk, respectively, and incubated for 48 h. During this time cells were stimulated with camptothecin (1 μm) or staurosporine (1 μm) for 6 h. Cells were harvested and washed once in Krebs Ringer-buffer (145 mm NaCl, 5 mm KCl, 3 mm CaCl2, 10 mm glutamate, 1.2 mm NaH2PO4, 1 mmMgSO4, and 20 mm Hepes buffer), stained with phosphatidylethanolamine labeled anti-mouse H2-Kkmonoclonal antibody (1:200 dilution, Pharmingen, San Diego, CA) to identify transfected cells and fluorescein isothiocyanate-labeled annexin V (1:200 dilution, Alexis Biochemicals) for 30 min on ice and analyzed by FACS-Scan. Rates of apoptosis in transfected Jurkat cells were determined by evaluation of the percentage of annexin-positive cells within the H2-Kk-positive population by flow cytometry. 2 × 107 Jurkat TAg cells per assay point were transiently co-transfected with 10 μg of epitope-tagged reporter kinase constructs plus 30 μg of the various sIgG cDNA expression plasmids, encoding sIgG-receptor chimera or receptor control as indicated (sIgG-receptor expression plasmid was used in 3-fold molar excess to statistically ensure cotransfection in each cell, which harbors the reporter kinase construct). 48 h posttransfection (with or without stimulation with 50 ng/ml PMA and 1 μg/ml ionomycin for 15 min), cells were harvested, solubilized in lysis buffer (5 mm NaP2P, 5 mm NaF, 5 mm EDTA, 50 mm NaCl, 50 mm Tris, pH 7.3, 2% Nonidet P-40, and 50 μg/ml each aprotinin and leupeptin) for 30 min on ice and centrifuged at 16,000 × g for 15 min at 4 °C. After preclearing, the epitope-tagged protein kinases were immunoprecipitated by incubation overnight at 4 °C with 2 μg of the respective anti-tag antibody and protein-G-Sepharose (Pharmacia Biotech Inc.) for 1 h at 4 °C. The immunoprecipitates were washed six times with lysis buffer. The reporter protein kinase was then resolved by immunoblotting with anti-active and pan mitogen-activated protein kinase (ERK and JNK) antibodies (New England Biolabs, Inc.). A representative result of at least five experiments is shown. 2 × 107 Jurkat TAg cells per assay point were co-transfected with 1 μg HA-tagged JNK constructs plus 20 μg of the sIgG-receptor chimera or receptor control as indicated. 40 h posttranfection cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 60 min and seeded on poly-l-lysine-coated glass cover slips and incubated at 37 °C and for 10 min. Cover slips were washed, and the cells fixed and permeabilized with the Fix and Perm Kit (An der Grub Bioresearch), according to the manufacturer's instructions. Rhodamine-conjugated rabbit anti-human IgG-Fc and fluorescein conjugated rat anti-HA antibodies were added to the permeabilization solution, and cells incubated for 20 min at 25 °C. The cover slips were washed four times with PBS and mounted on slides with mowiol mounting solution (Calbiochem). Microscopy was performed with the Olympus BX50 Fluorescence Microscope, and pictures were taken with the digital camera Microview TE/CCD1317-K/1 (Princeton Scientific Instruments) using the metamorph imaging software (Universal Imaging Corporation). Finally the pictures were processed with the AutoDeblur 7.5 software (Autoquant Imaging, Inc.). 2 × 107 Jurkat TAg cells per assay point were transfected with 30 μg of cDNA expression plasmids encoding chimeric sIgG-receptors, sIgG or sIgG-LRP respectively, plus 1 μg of JNK-2-HA for subcellular fractionation of JNK. After incubation for 44–46 h cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for up to 60 min at 37 °C or left unstimulated (Me2SO buffer control). Cell fractionation was performed by subsequent lysis and centrifugation in equivalent amounts of different buffers: "n, nuclear fraction" (pelleted by 10 min and 1000 × g centrifugation) and "s, cytoplasmic (post-1000 × g soluble) fraction" were resolved by lysis buffer without detergent (5 mmNa3VO4, 5 mm NaF, 2 mmEDTA, 5 mm EGTA, 1 mm dithiothreitol, 20 mm Tris pH 7.3, and 50 μg/ml each aprotinin and leupeptin), "pt, particulate/membrane fraction" was resolved in lysis buffer containing 1% Nonidet P-40, as described (29Bauer B. Krumbock N. Ghaffari-Tabrizi N. Kampfer S. Villunger A. Wilda M. Hameister H. Utermann G. Leitges M. Uberall F. Baier G. Eur. J. Immunol. 2000; 30: 3645-3654Crossref PubMed Scopus (48) Google Scholar). Data are expressed as the means ± S.E. (n = 6). Two distinct sIgG-receptor chimeric expression constructs, termed sIgG-LRP and sIgG-APP, were constructed by fusing transmembrane region and extracellular IgG-Fc domain to the cytoplasmic COOH termini of the human LRP and APP cDNA, respectively (see Fig. 1 A for illustration). Ectopic Jurkat TAg cell transfection with these chimeric sIgG constructs (and the truncated sIgG-receptor control protein, expressing no cytoplasmic fusion tail) followed by FACS employing the anti-IgG Fc antibody demonstrated cell surface expression of sIgG chimera in transfected cells (Fig. 1 B). Interestingly (and most likely due to altered kinetics of processing, surface expression and/or endocytotic rates exerted by the internalization signals of the LRP and APP COOH-tail domains), reduced levels of surface expression of these chimeric sIgG-receptorsversus sIgG-receptor control could be reproducibly detected (48 h posttransfection, 20% versus50% sIgG-receptor-expressing cells, respectively). Immunoblot analysis of plasma membrane fractions employing Fc-specific antisera detected protein smears of 54–65 kDa proteins in cells transfected with the corresponding sIgG-expression plasmids but not in cells transfected with empty vector control (Fig. 1 C). The distinct molecular appearance of the different sIgG-receptor proteins indicates posttranslational glycoyslation of the Fcdomain. First, and for comparison purposes with our chimeric sIgG-LRP receptor, we have examined the ability of endogenous LRP signaling pathways to modulate cellular survival. Activated α2-macroglobulin, an established ligand of LRP (1Herz J. Neuron. 2001; 29: 571-581Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) reduces (by 40%) apoptosis of the ex vivocultured rat cerebellar granule cells (Fig.2 A). Native α2-macroglobulin, which is not a ligand for LRP, served as control. Similarly, decreased susceptibility to DNA damage, were reproducibly observed by sIgG-LRP expression (29% increase in survival, relative to sIgG control; Fig. 2 B). As positive control, transient bcl-2 overexpression also inhibited DNA damage-induced apoptosis in parallel experiments (Fig. 2 C). This provides evidence that α2-macroglobulin-exerted survival effects (through the endogenous LRP-mediated pathway) can be bypassed by ectopic sIgG-LRP expression. We then extended our analysis by testing the signaling role of endogenous LRP on mitogenic signaling function employing transient transfection with promoter reporter constructs. Activated α2-macroglobulin by itself did not affect signals to Elk-1. Interestingly, however, α2-macroglobulin treatment altered the cellular response to NGF, e.g. significantly abrogated mitogen-induced Elk-1 and cJun transcriptional activity (Fig.3 and not shown), indicating that the ERK and/or JNK pathways operate downstream of LRP in the regulation of NGF signaling. This very reproducible result prompted us to examine mitogenic signaling modulation employing our sIgG-LRP chimeric receptor approach. To investigate the potential role of sIgG-LRP and sIgG-APP (in comparison to sIgG-control) in induction of the Elk-1, cJun, and NF-κB-promoter reporters, cells were transiently co-transfected with sIgG-chimeric cDNA constructs and the promoter reporters and subsequently stimulated for up to 16 h with PMA/ionomycin or left unstimulated, lysed, and assayed for reporter gene expression. Consistent to the endogenous LRP function (Fig. 3), sIgG-LRP, and sIgG-APP by itself (with or without bead-bound anti-Fc antibody-mediated clustering) are not sufficient to induce signaling function on these pathways under investigation (Fig.4 A). Additionally, no change in basal phosphotyrosine staining of total cellular proteins could be detected upon expression of sIgG-APP and sIgG-LRP (with or without anti-Fc cross-linking, not shown). However, expression of the established receptor sIgG-Syk, a protein tyrosine kinase isoenzyme of the ZAP-70 family, induced strong changes of total cellular phosphotyrosine protein staining (not shown), strictly dependent on anti-Fc cross-linking (26Zeitlmann L. Knorr T. Knoll M. Romeo C. Sirim P. Kolanus W. J. Biol. Chem. 1998; 273: 15445-15452Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Therefore we decided to further investigate the cellular signaling cascade(s) involving sIgG-APP and sIgG-LRP vis-à-vis of mitogenic activation leading to Elk-1, c-Jun, and NF-κB-transactivation employing pleiotropic phorbol ester and ionomycin stimulation. As shown in Fig. 4 B, transient overexpression of sIgG-LRP (relative to the sIgG-receptor control) caused a significant decrease in the transcriptional activation of the Elk-1 (Fig. 4, B and C) and cJun (Fig.4 D) promoter reporter gene, (with or without bead-bound anti-Fc antibody-mediated clustering) indicating interference function(s) of LRP on the given signaling pathways. This phenomenon was also observed with endogenous LRP signaling (see Fig.3). Interestingly, and in contrast to similar binding results of JIP to both, LRP and APP (1Herz J. Neuron. 2001; 29: 571-581Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), sIgG-APP mutant was much less effective (cJun, Fig. 4 D) and even failed (Elk-1, Fig. 4, Band C) to significantly abrogate mitogen-induced promoter reporter activity even though both transgenes have been properly expressed (not shown an
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