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• W2049520040 abstract "Grb4 is an adaptor protein consisting of three src homology (SH) 3 domains and a single SH2 domain. We previously cloned Grb4 as a direct interacting partner of Bcr-Abl and v-Abl via the Grb4 SH2 domain. We now show that overexpression of Grb4 results in significant inhibition of v-Abl-induced transcriptional activation from promitogenic enhancer elements such as activator protein 1 (AP-1) and serum-responsive element (SRE). We demonstrate that the inhibitory activity of Grb4 is independent of the direct interaction of v-Abl and Grb4: a Grb4 mutant that lacks a functional SH2 domain shows an even more pronounced inhibition of AP-1/SRE. Further mutational analysis revealed that the first two SH3 domains primarily mediate the inhibitory function. The inhibitory activity of Grb4 is specific for c-jun/c-fos-regulated promoter elements and is located downstream of MEKK1 and JNK because co-expression of Grb4 resulted in down-regulation of MEKK1-induced AP-1 activity without affecting JNK activity. Thus, the nuclear pool of Grb4 is likely to mediate this inhibition. Indeed, cell fractionation and fluorescence microscopy studies revealed that the stronger inhibitory potential of the Grb4 SH2 mutant occurred in conjunction with increased nuclear localization of this mutant. Our results suggest a novel role for Grb4 in the inhibition of promitogenic enhancer elements such as 12-O-tetradecanoylphorbol-13-acetate-responsive element and SRE. Grb4 is an adaptor protein consisting of three src homology (SH) 3 domains and a single SH2 domain. We previously cloned Grb4 as a direct interacting partner of Bcr-Abl and v-Abl via the Grb4 SH2 domain. We now show that overexpression of Grb4 results in significant inhibition of v-Abl-induced transcriptional activation from promitogenic enhancer elements such as activator protein 1 (AP-1) and serum-responsive element (SRE). We demonstrate that the inhibitory activity of Grb4 is independent of the direct interaction of v-Abl and Grb4: a Grb4 mutant that lacks a functional SH2 domain shows an even more pronounced inhibition of AP-1/SRE. Further mutational analysis revealed that the first two SH3 domains primarily mediate the inhibitory function. The inhibitory activity of Grb4 is specific for c-jun/c-fos-regulated promoter elements and is located downstream of MEKK1 and JNK because co-expression of Grb4 resulted in down-regulation of MEKK1-induced AP-1 activity without affecting JNK activity. Thus, the nuclear pool of Grb4 is likely to mediate this inhibition. Indeed, cell fractionation and fluorescence microscopy studies revealed that the stronger inhibitory potential of the Grb4 SH2 mutant occurred in conjunction with increased nuclear localization of this mutant. Our results suggest a novel role for Grb4 in the inhibition of promitogenic enhancer elements such as 12-O-tetradecanoylphorbol-13-acetate-responsive element and SRE. src homology mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase c-Jun N-terminal kinase activator protein 1 serum-responsive element 12-O-tetradecanoylphorbol-13-acetate TPA-responsive element polyacrylamide gel electrophoresis β-galactosidase wild-type heterogeneous nuclear ribonucleoprotein Grb4 (also referred to as Nckβ or Nck-2) belongs to an emerging class of adaptor proteins that consist of functional src homology (SH)1 domains but lack intrinsic catalytic activity (1Birge R.B. Knudsen B.S. Besser D. Hanafusa H. Genes Cells. 1996; 1: 595-613Crossref PubMed Scopus (122) Google Scholar, 2Chen M. She H. Davis E.M. Spicer C.M. Kim L. Ren R. Le Beau M.M. Li W. J. Biol. Chem. 1998; 273: 25171-25178Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 3Braverman L.E. Quilliam L.A. J. Biol. Chem. 1999; 274: 5542-5549Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 4Tu Y. Li F. Wu C. Mol. Biol. Cell. 1998; 9: 3367-3382Crossref PubMed Scopus (161) Google Scholar, 5Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). The Nck family of proteins share a common structure consisting of three SH3 domains and a C-terminal SH2 domain. SH3 domains are known to mediate interactions with proline-rich motifs; SH2 domains bind to specific phosphorylated tyrosine residues. Grb4 exhibits high homology to Nck (Nckα). For Nck, a functional role in JNK activation (6Stein E. Huynh-Do U. Lane A.A. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1998; 273: 1303-1308Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) has been proposed via Nck-interacting kinase/Ste20 kinase (7Su Y.C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (219) Google Scholar, 8Becker E. Huynh-Do U. Holland S. Pawson T. Daniel T.O. Skolnik E.Y. Mol. Cell. Biol. 2000; 20: 1537-1545Crossref PubMed Scopus (117) Google Scholar). Nck-interacting kinase has been reported to bind Nck and MEKK1 and activate the JNK/stress-activated protein kinase pathway (7Su Y.C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (219) Google Scholar, 9Su Y.C. Treisman J.E. Skolnik E.Y. Genes Dev. 1998; 12: 2371-2380Crossref PubMed Scopus (126) Google Scholar). Nck has also been identified as an interaction partner of Sos (10Hu Q. Milfay D. Williams L.T. Mol. Cell. Biol. 1995; 15: 1169-1174Crossref PubMed Google Scholar), Cbl (11Rivero-Lezcano O.M. Sameshima J.H. Marcilla A. Robbins K.C. J. Biol. Chem. 1994; 269: 17363-17366Abstract Full Text PDF PubMed Google Scholar), WASP (12Rivero-Lezcano O.M. Marcilla A. Sameshima J.H. Robbins K.C. Mol. Cell. Biol. 1995; 15: 5725-5731Crossref PubMed Scopus (280) Google Scholar), and PAK (13Bokoch G.M. Wang Y. Bohl B.P. Sells M.A. Quilliam L.A. Knaus U.G. J. Biol. Chem. 1996; 271: 25746-25749Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 14Galisteo M.L. Chernoff J. Su Y.C. Skolnik E.Y. Schlessinger J. J. Biol. Chem. 1996; 271: 20997-21000Abstract Full Text PDF PubMed Scopus (237) Google Scholar). Proteins reported to interact with Grb4 include PAK and Sos1 (3Braverman L.E. Quilliam L.A. J. Biol. Chem. 1999; 274: 5542-5549Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Nck has been shown to transform fibroblasts (15Chou M.M. Fajardo J.E. Hanafusa H. Mol. Cell. Biol. 1992; 12: 5834-5842Crossref PubMed Scopus (81) Google Scholar), whereas at this time, there are somewhat contradictory reports regarding the function of Grb4 on mitogenesis. Grb4 has been shown to cooperate with v-Abl to transform NIH3T3 fibroblasts and synergistically activate the Elk-1 pathway (3Braverman L.E. Quilliam L.A. J. Biol. Chem. 1999; 274: 5542-5549Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Another group demonstrated that Grb4 is a potent inhibitor of epidermal growth factor-stimulated and platelet-derived growth factor-stimulated DNA synthesis (2Chen M. She H. Davis E.M. Spicer C.M. Kim L. Ren R. Le Beau M.M. Li W. J. Biol. Chem. 1998; 273: 25171-25178Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We previously identified Grb4 as a direct interaction partner of Bcr-Abl (5Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). This interaction resulted in a significant redistribution of both proteins, most likely involving actin reorganization. Other reports also describe an involvement of Grb4 in cytoskeletal reorganization. Tu et al. (4Tu Y. Li F. Wu C. Mol. Biol. Cell. 1998; 9: 3367-3382Crossref PubMed Scopus (161) Google Scholar) showed that Grb4 interacts with PINCH, a molecule that links growth factor receptors with integrin signaling. Recently, Chen et al. (16Chen M. She H. Kim A. Woodley D.T. Li W. Mol. Cell. Biol. 2000; 20: 7867-7880Crossref PubMed Scopus (75) Google Scholar) showed that Grb4 but not Nck blocks Rac-1-mediated membrane ruffling and formation of lamellipodia. Thus, despite the high homology between Grb4 and Nck, the functional characteristics of both proteins seem to be quite different. In this report, we show that Grb4 inhibits the transcription from promitogenic promoters such as AP-1 and SRE. AP-1 acts as a dimeric DNA sequence-specific transcriptional activator composed of homo- or heterodimers of Jun, Fos, and/or ATF. Jun homodimers and Jun-Fos heterodimers preferentially recognize the TPA-responsive element, whereas Jun-ATF and ATF-ATF dimers bind to the cyclic AMP-responsive element. Activation of AP-1 has been reported to be induced after a variety of stimuli (including TPA, growth factors, cytokines, T-cell activation, neurotransmitters, and UV radiation) and to play a role in cell proliferation and oncogenic transformation (for a review, see Refs. 17Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3279) Google Scholar and 18Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar). AP-1 activity is generally regulated by the expression level of AP-1 proteins and their activity state (19Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2258) Google Scholar). AP-1 proteins have been reported to control their own expression level. Transcription of c-Jun is usually mediated through the TPA-responsive element (TRE), which is recognized by AP-1. Transcription of c-Fos is regulated by the SRE. In addition, secondary modificationse.g. modifications (e.g. phosphorylation on serine and threonine residues) enhance the stability of these proteins (20Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (847) Google Scholar, 21Tsurumi C. Ishida N. Tamura T. Kakizuka A. Nishida E. Okumura E. Kishimoto T. Inagaki M. Okazaki K. Sagata N. et al.Mol. Cell. Biol. 1995; 15: 5682-5687Crossref PubMed Scopus (128) Google Scholar). Serine phosphorylation of c-Jun also results in increased transcriptional activity (22Smeal T. Hibi M. Karin M. EMBO J. 1994; 13: 6006-6010Crossref PubMed Scopus (85) Google Scholar). A co-activator designated JAB1 has been identified, and it stabilizes AP-1-DNA complexes by interaction with c-Jun or JunD (23Claret F.X. Hibi M. Dhut S. Toda T. Karin M. Nature. 1996; 383: 453-457Crossref PubMed Scopus (409) Google Scholar). Known inhibitors of AP-1 activity include ligands of nuclear receptors (e.g. glucocorticoid receptor), which are thought to reduce the interaction between c-Jun and the transcriptional co-activator CBP/p300 (24Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1928) Google Scholar), and the interferon-inducible p202, which interacts directly with c-Jun and c-Fos (25Min W. Ghosh S. Lengyel P. Mol. Cell. Biol. 1996; 16: 359-368Crossref PubMed Scopus (142) Google Scholar). Another model proposed for the inhibition of AP-1 by ligand-activated nuclear receptors involves blocking of c-Jun phosphorylation (26Caelles C. Gonzalez-Sancho J.M. Munoz A. Genes Dev. 1997; 11: 3351-3364Crossref PubMed Scopus (292) Google Scholar). The oncogene v-Abl has been shown to activate the serum-responsive and TPA-responsive elements (27Hori Y. Kaibuchi K. Fukumoto Y. Oku N. Takai Y. Oncogene. 1990; 5: 1201-1206PubMed Google Scholar), which is correlated with its promitogenic potential. It could be demonstrated that v-Abl-induced activation of TPA-responsive elements is inhibited by dominant negative Rac-1 but not Ras, suggesting that Rac-1 is involved in the signal transduction pathway leading to activation of AP-1 (28Renshaw M.W. Lea-Chou E. Wang J.Y. Curr. Biol. 1996; 6: 76-83Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In addition, dominant negative Rac also inhibited v-Abl-induced activation of SRE and other mitogenic responses such as JNK1, mitogen-activated protein kinase, and extracellular signal-regulated kinase 2 activation (28Renshaw M.W. Lea-Chou E. Wang J.Y. Curr. Biol. 1996; 6: 76-83Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Searching for a functional role for Grb4 in Abl-mediated signaling, we found that Grb4 is a potent inhibitor of v-Abl-induced activation of TRE and SRE. AP-1 inhibition by various Grb4 mutants with different subcellular localization strongly suggested that the nuclear pool of Grb4 mediates this inhibition. The anti-xpress™ antibody was purchased from Invitrogen (Groningen, The Netherlands). Anti-Abl antibodies were obtained from Pharmingen (8E9) and Calbiochem-Novabiochem (Ab3) (Schwalbach, Germany), anti-c-Jun (H-79) and anti p-c-Jun (KM-1) and anti-p-JNK (G-7) antibodies were obtained from Santa Cruz (Heidelberg, Germany). The Grb4 mutants were generated using polymerase chain reaction-based mutagenesis. For mutation of the SH2 domain, arginine within the FLVR motif was changed to leucine. SH3 domains were mutated by exchange of the characteristic tryptophan with lysine (29Tanaka M. Gupta R. Mayer B.J. Mol. Cell. Biol. 1995; 15: 6829-6837Crossref PubMed Scopus (221) Google Scholar). EYFP and ECFP mutants of EGFP and the EY(C)FP-Grb4 fusion constructs were obtained as described previously (5Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). 293, NIH3T3 and COS7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. COS7 cells were transfected using Gene Porter (GTS Inc., Biozol GmbH, Eching, Germany) according to manufacturer's recommendations. NIH3T3 cells were transfected using Gene Porter or Superfect™ (Qiagen, Hilden, Germany). 293 cells were transfected usingN-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate (Roche Molecular Biochemicals). Immunoprecipitations and immunoblotting were done as described previously (30Duyster J. Baskaran R. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1555-1559Crossref PubMed Scopus (106) Google Scholar). Briefly, cells were harvested in cold phosphate-buffered saline containing 1 mm sodium vanadate, pelleted, and lysed in lysis buffer containing 10 mmTris-HCl (pH 7.4), 5 mm EDTA, 130 mm NaCl, and 1% Triton. Proteinase inhibitor mixture Complete™ tablets were added according to the manufacturer's recommendations (Roche Molecular Biochemicals). After clarification by centrifugation and preclearing with protein A-Sepharose, antibody-protein complexes were brought down with 30 μl of protein A-Sepharose (Amersham Pharmacia Biotech). Lysates and bound fractions of immunoprecipitations were subjected to SDS-PAGE, and blotting was performed on polyvinylidene difluoride membranes (Immobilon-P; Millipore GmbH, Eschborn, Germany). AP-1 activation was determined as described previously (5Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). Briefly, in 293 cells or NIH3T3 cells, 1 μg of the AP-1/luciferase reporter construct, which contains a basic TATA promoter joined to tandem repeats of AP-1 or SRE binding elements (Stratagene, La Jolla, CA), 100 ng of a plasmid carrying the β-GAL gene, and tagged Grb4 constructs as indicated were co-transfected with 1 μg of pCDNA3.1/v-Abl. Cells were cultured for 3 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, and luciferase activity was measured using a luciferase assay kit (Promega, Mannheim, Germany) and a luminometer (Berthold, Pforzheim, Germany). For transforming growth factor β-mediated SMAD activation, 1 μg of a SMAD-dependent luciferase reporter construct was used instead of the AP-1 construct. Nuclear and cytosolic extracts were prepared as follows: transiently transfected 293 cells were pelleted, washed once in ice-cold phosphate-buffered saline, and lysed in buffer A (10 mm HEPES, pH 8, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 300 mm sucrose, 0.1% Nonidet P-40, and a proteinase inhibitor mixture (Complete™; Roche Molecular Biochemicals). After a short centrifugation, the cytosolic supernatant was removed, the nuclear pellet was again resuspended in buffer A, and extraction was repeated. Nuclear proteins were extracted by resuspending the nuclear pellet in buffer B (20 mm HEPES, pH 8, 20% glycerol, 100 mm KCl, 100 mm NaCl, 0.2 mm EDTA, 0.5 mm dithiothreitol, and a proteinase inhibitor mixture). After 10 s of sonification and a pulse centrifugation, the supernatant containing the nuclear fraction was frozen. For shift and supershift assays, 3 μg of protein was used and incubated with the33P-labeled AP-1 probe at room temperature for 30 min. Antibodies (4 μg) were added as indicated. Complexes were separated by nondenaturing PAGE (8%). Gels were dried, and DNA-protein complexes were visualized by autoradiography. Fixation and detection of EGFP fusion constructs in fixed cells was done as described previously (5Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). Briefly, COS7 cells were cultured and transfected on gelatin-coated chamber slides (Nunc GmbH, Wiesbaden, Germany). 72 h after transfection, cells were starved for at least 6 h, washed with cold phosphate-buffered saline containing 1 mm vanadate, and fixed with 3.7% paraformaldehyde for 15 min. Cells were then washed twice with phosphate-buffered saline, overlaid with mounting medium (Molecular Probes Europe BV, Leiden, The Netherlands), covered with a coverslide, and visualized using a fluorescence microscope (Olympus Optical Co. GmbH, Hamburg, Germany) connected to a digital imaging system (T.I.L.L. Photonics, Munich, Germany). For detection of ECFP and EYFP fluorochromes, the appropriate filter sets were used (Chroma; AHF AG, Tuebingen, Germany). The Mig-IRES-v-abl construct was cloned by ligating v-abl cDNA (a kind gift from J. Wang, University of California San Diego, San Diego, CA) cut withBamHI/HindIII together with the ECMV-IRES sequence cut with EcoRI/BamHI into the MigRI vector (a kind gift from W. Pear, University of Pennsylvania Medical Center, Philadelphia, PA) cut with EcoRI andHindIII. The FLAG epitope-tagged Grb4-SH2 mutant cDNA was digested from the pcDNA3.1-Zeo vector (Invitrogen, Karlsruhe, Germany) with PmeI and cloned into the HpaI site of the Mig-IRES-v-abl construct, generating Mig-Grb4SH2-v-abl. Viral particles were produced by transiently transfecting the ecotropic Phoenix cell line (kindly supplied by G. Nolan, Stanford University School of Medicine, Standford, CA) with the different constructs, as described previously (31Bai R.Y. Ouyang T. Miething C. Morris S.W. Peschel C. Duyster J. Blood. 2000; 96: 4319-4327Crossref PubMed Google Scholar). 105 NIH3T3 cells were infected in 6-well plates in the presence of 4 μg/ml polybrene overnight. After 2 days, cells were analyzed for v-abl expression by intracellular staining for Abl, as described previously (32Hao S.X. Ren R. Mol. Cell. Biol. 2000; 20: 1149-1161Crossref PubMed Scopus (127) Google Scholar). Equal numbers of infected cells were plated in duplicate in soft agar medium according to procedures described previously (31Bai R.Y. Ouyang T. Miething C. Morris S.W. Peschel C. Duyster J. Blood. 2000; 96: 4319-4327Crossref PubMed Google Scholar). The plates were monitored for colony growth, and photographs were taken after 20 days of culture. We previously identified Grb4 as a specific interaction partner of Bcr-Abl binding to the Abl portion of Bcr-Abl (5Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). Glutathione S-transferase binding assays revealed that this interaction is mediated by the SH3 as well as the SH2 domains of Grb4 and that Grb4 was able to bind to v-Abl in vitro (5Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). To investigate the role of the SH2 and SH3 domains for complex formation of Grb4 and v-Abl in vivo in more detail, we created a Grb4 SH2 mutant (Grb4SH2) and a Grb4 mutant with loss of function mutations in all three SH3 domains (Grb4SH3-1,2,3) of Grb4 (29Tanaka M. Gupta R. Mayer B.J. Mol. Cell. Biol. 1995; 15: 6829-6837Crossref PubMed Scopus (221) Google Scholar). In a third mutant, SH2 and SH3 mutations were combined (Grb4SH2/SH3-1,2,3) (TableI). Co-immunoprecipitation experiments revealed that co-expression of Grb4 and v-Abl resulted in complex formation between v-Abl and Grb4 independent of the kinase activity of v-Abl (Fig. 1 A, top panel, lanes 1 and 5). The complex between Grb4 and kinase-defective v-Abl (v-AblKD) was dependent on the SH3 domains but independent of the SH2 domain of Grb4 (Fig. 1 A, top panel, lanes 2–4). In contrast, complex formation between Grb4 and kinase-active v-Abl was significantly reduced by a mutation in the SH2 domain of Grb4 (Fig.1 A, top panel, lane 7). Mutation of the SH3 domains had little effect on complex formation with kinase-active v-Abl (Fig.1 A, top panel, lane 6). A similar result was obtained when we analyzed complex formation with the other oncogenic variant of Abl, Bcr-Abl (Fig. 1 B). Both kinase-defective Bcr-Abl (Bcr-AblKD) and kinase-active Bcr-Abl can co-immunoprecipitate Grb4 (Fig. 1 B, top panel, lanes 1 and 2). According to the data obtained with v-abl, the complex between Grb4 and Bcr-Abl was also abrogated by a mutation in the SH2 domain of Grb4 (Fig. 1 B, top panel, lane 3). Similar to v-Abl, kinase-defective Bcr-Abl binds to Grb4 independent of the SH2 domain but dependent on the SH3 domains (data not shown). Thus, in vivo complex formation between Grb4 and kinase-active v-Abl or Bcr-Abl is mediated mainly by the SH2 domain of Grb4. Interestingly, the SH3 domain only contributes to the complex of kinase-defective v-Abl and Grb4, suggesting that the SH3-mediated binding of Grb4 to v-Abl might be negatively regulated by phosphorylation.Table IGrb4 and Grb4 mutants Open table in a new tab Figure 1Complex formation between v-Abl and Grb4. A, kinase-defective v-Abl (KD) or v-Abl was co-expressed with either tagged (xpress™) Grb4, the triple SH3 Grb4 mutant (Grb4SH3–1,2,3), the SH2 mutant (Grb4SH2), or the combined mutant (Grb4SH2/SH3–1,2,3) in COS1 cells. The Grb4 mutants were immunoprecipitated (IP) using anti-xpress™ antibody (first and third panels) or nonspecific rabbit anti-mouse antibody (RαM) as control (second panel). Immunoprecipitations were analyzed by αAbl antibody (first and second panel) or anti-xpress™ antibody (third panel) to demonstrate immunoprecipitation of Grb4. Lysates analyzed by αAbl antibody demonstrate equal expression of v-Abl in all samples (fourth panel). B,kinase-defective Bcr-Abl (KD) was co-expressed with tagged Grb4 WT (lane 1), and Bcr-Abl was co-expressed with either Grb4 or Grb4SH2 (lanes 2 and 3). The Grb4 proteins were immunoprecipitated using αexpress antibody (first and third panels) from lysates containing equal amounts of Bcr-Abl (fourth panel). Control immunoprecipitations were performed using equal amounts of rabbit anti-mouse antibody (RαM) (second panel). Immunoprecipitates were analyzed by Western blotting using the antibodies indicated.View Large Image Figure ViewerDownload (PPT) We have previously shown that co-expression of Grb4 with v-Abl resulted in inhibition of v-Abl-induced activation of an AP-1 reporter construct in a concentration-dependent manner (5Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). We were interested in discovering whether disruption of the direct interaction between v-Abl and Grb4 would have an effect on the Grb4-mediated AP-1 inhibition. Surprisingly, co-expression of v-Abl and the Grb4SH2 mutant that no longer binds to v-Abl caused an even stronger inhibition of v-Abl-mediated activation of an AP-1 reporter construct (Fig.2 A). Co-expression of v-Abl and Grb4SH2 resulted in about 30% more inhibition of AP-1 activity compared with that induced by the same amount of wild-type (WT) DNA (Fig. 2 A, compare bars 2 and 5). Western blot analysis of the lysates showed that the expression level of the Grb4SH2 mutant was even lower than that of WT Grb4 (Fig.2 B). In turn, compared with WT Grb4, the same extent of Grb4SH2-induced inhibition was obtained when only a third of the WT Grb4 DNA was transfected (76% versus 79% inhibition; Fig.2 A, compare bars 4 and 5). Therefore, the difference in the inhibitory activity is not due to the expression level of both proteins but is specific for the introduced mutation. All luciferase activities were normalized by β-GAL activity. β-GAL values after co-expression of a vector carrrying the β-GAL gene were similar and were not influenced by Grb4 co-expression (see Fig.3 C). In addition, in vitro translation of v-Abl in the presence or in the absence of Grb4 did not change the autophosphorylation capacity of v-Abl (data not shown). Therefore, we concluded that the inhibitory activity of Grb4 upon AP-1 activity is independent of the direct interaction of v-Abl and Grb4 and is located downstream of v-Abl.Figure 3Grb4-mediated inhibition of MEKK1-induced AP-1 activity. A, 293 cells were co-transfected with the AP-1 reporter construct (1 μg), a plasmid containing the β-GAL gene (100 ng), constitutively active MEKK1 (50 ng), and tagged Grb4 constructs as indicated. Luciferase activity was normalized for transfection efficiency using β-GAL values. B, 293 cells were co-transfected with the SRE reporter construct (1 μg), a plasmid containing the β-GAL gene (100 ng), constitutively active MEKK1 (25 ng), and tagged Grb4 constructs (1 μg) or vector control (Ø; 1 μg). Luciferase activity was normalized for transfection efficiency using β-GAL values. C, 293 cells were co-transfected with a plasmid containing the β-GAL gene (100 ng), constitutively active MEKK1 (25 ng), and tagged Grb4 constructs (1 μg) or vector control (Ø; 1 μg). D, 293 cells were co-transfected with the SMAD-dependent luciferase reporter construct (1 μg), a plasmid containing the β-GAL gene (100 ng), constitutively active MEKK1 (25 ng), and tagged Grb4 constructs (1 μg) or vector control (Ø; 1 μg). Luciferase activity was normalized for transfection efficiency using β-GAL values. Data represent the relative luciferase activity of three independent experiments.View Large Image Figure ViewerDownload (PPT) To further define the target in the signaling cascade of Grb4-mediated AP-1 inhibition, we tested whether MEKK1-induced AP-1 activity is affected by Grb4. Similar to v-Abl, co-expression of constitutively active MEKK1 and Grb4 resulted in inhibition of MEKK1-induced AP-1 activation in a concentration-dependent manner (Fig. 3 A). The SH2 mutant of Grb4 again exhibited significantly increased inhibition. The same result was obtained when we measured the effect of Grb4 on MEKK1-induced SRE activation using a different reporter construct (Fig.3 B). These results confirmed the data obtained with v-Abl. In addition, the inhibitory effect of Grb4 is also demonstrable with another promitogenic enhancer element such as SRE. Importantly, Grb4 did not show any detectable suppression of β-GAL activity (Fig.3 C) or of transforming growth factor β-inducible activation of a SMAD-responsive reporter construct (Fig.3 D). Thus, Grb4 specifically inhibits the activation of promitogenic reporter systems such as SRE and TRE downstream or at the same level of MEKK1. Grb4 consists of one C-terminal SH2 domain and three SH3 domains (Table I). Our data show that disruption of the SH2 domain even enhances the inhibitory effect on transription from TRE/SRE-regulated reporter constructs. To investigate the role of the Grb4 SH3 domains for the inhibition of these promitogenic enhancer elements, we created three additional single SH3 mutants of Grb4SH2 (Grb4SH2/SH3-1, Grb4SH2/SH3-2, and Grb4SH2/SH3-3) and a combination thereof by changing the central tryptophan to lysine within each SH3 domain (Table I) (29Tanaka M. Gupta R. Mayer B.J. Mol. Cell. Biol. 1995; 15: 6829-6837Crossref PubMed Scopus (221) Google Scholar). These mutants were co-expressed with MEKK1 and analyzed for their ability to down-regulate AP-1-dependent luciferase activity (Fig.4). Compared with the Grb4SH2 mutant, the triple SH3 mutant (Grb4SH2/SH3-1,2,3) failed to inhibit MEKK1-mediated AP-1 activation (Fig. 4, compare bars 2 and 6). When the single SH3 mutants in combination with the SH2 mutation were co-expressed with MEKK1, mutation of the first and the second SH3 domains resulted in significantly weaker inhibition compared with that of the mutation affecting the third SH3 domain (Fig. 4, comparebars 3, 4, and 5). Similar results were obtained for v-Abl-induced AP-1 activity (data not shown). These findings suggest that the first two SH3 domains of Grb4 mediate the inhibition of transcription from promitogenic enhancer elements such as TRE in this assay. JNK is a direct effector of MEKK1 (33Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). JNK regulates the transcriptional activation domain of c-jun for both the SRE and TRE enhancer elements. Therefore, we wished to determine the activity of JNK because it is located further downstream within the AP-1 signaling cascade. To test whether the inhibition of MEKK1-induced AP-1 activation is due to a decrease in JNK activity, we measured JNK activation and AP-1-dependent luciferase activity within the same cells (Fig. 5). MEKK1-in" @default.
• W2049520040 created "2016-06-24" @default.
• W2049520040 creator A5062556043 @default.
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