Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and itsDrosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK. Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and itsDrosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK. Eph family receptor tyrosine kinases transmit signals that direct cell migration, cell targeting, and cell-cell aggregation (1Pandey A. Shao H. Marks R.M. Polverini P.J. Dixit V.M. Science. 1995; 268: 567-569Crossref PubMed Scopus (344) Google Scholar, 2Drescher U. Kremoser C. Handwerker C. Loschinger J. Noda M. Bonhoeffer F. Cell. 1995; 82: 359-370Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 3Orioli D. Klein R. Trends Genet. 1997; 13: 354-359Abstract Full Text PDF PubMed Scopus (155) Google Scholar, 4Bohme B. VandenBos T. Cerretti D.P. Park L.S. Holtrich U. Ruebsamen-Waigmann H. Strebhardt K. J. Biol. Chem. 1996; 271: 24747-24752Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 5Wang H.U. Anderson D.J. Neuron. 1997; 18: 383-396Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). These receptors are functionally subdivided into two subclasses (EphA or EphB) based on their overlapping affinities for either glycerolphosphatidylinositol-linked (ephrins A1–A5) or transmembrane (ephrins B1–B3) protein ligands (6Eph Nomenclature Committee Cell. 1997; 90: 403-404Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 7Brambilla R. Klein R. Mol. Cell. Neurosci. 1995; 6: 487-495Crossref PubMed Scopus (78) Google Scholar). Many Eph family receptor tyrosine kinases and their ligands are reciprocally compartmentalized during development, consistent with their roles in directing migration and organization of specialized cell-cell interactions (2Drescher U. Kremoser C. Handwerker C. Loschinger J. Noda M. Bonhoeffer F. Cell. 1995; 82: 359-370Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 8Gale N.W. Holland S.J. Valenzuela D.M. Flenniken A. Pan L. Ryan T.E. Henkemeyer M. Strebhardt K. Hirai H. Wilkinson D.G. Pawson T. Davis S. Yancopoulos G.D. Neuron. 1996; 17: 9-19Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar, 9Bergemann A.D. Cheng H.-J. Brambilla R. Klein R. Flanagan J.G. Mol. Cell. Biol. 1995; 15: 4921-4929Crossref PubMed Scopus (138) Google Scholar, 10Cheng H. Nakamoto M. Bergemann A.D. Flanagan J.G. Cell. 1995; 82: 371-381Abstract Full Text PDF PubMed Scopus (666) Google Scholar). For example, tectal gradients of the EphA3 (Mek4) ligand, ephrin A2 (ELF-1), direct developmental targeting of retinal axons expressing EphA3 receptor (11Cheng H.-J. Flanagan J.G. Cell. 1994; 79: 157-168Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 12Nakamoto M. Cheng H.-J. Friedman G.C. McLaughlin T. Hansen M.J. Yoon C.H. O'Leary D.M. Flanagan J.G. Cell. 1996; 86: 755-766Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Similarly, axonal migratory paths of spinal motor neurons expressing Eph receptors, EphB4 and EphB3 (HTK/HEK2), are directed by segmental expression of the EphB4 ligand, ephrin B2 (5Wang H.U. Anderson D.J. Neuron. 1997; 18: 383-396Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). In a reconstituted system, 32D cells transfected with EphB3 (Hek2) receptors aggregate with cells transfected with the EphB3 ligand, ephrin B1 (4Bohme B. VandenBos T. Cerretti D.P. Park L.S. Holtrich U. Ruebsamen-Waigmann H. Strebhardt K. J. Biol. Chem. 1996; 271: 24747-24752Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The intracellular mediators of these targeting responses are incompletely defined. EphA2 (Eck) and EphB1 (ELK) are prototypic examples of these respective subclasses that signal through distinct cytoplasmic mediators. Ligand-activated EphA2 binds the p85 subunit of phosphatidylinositol 3-kinase and activates phosphatidylinositol 3-kinase (13Pandey A. Lazar D.F. Saltiel A.R. Dixit V.M. J. Biol. Chem. 1994; 269: 30154-30157Abstract Full Text PDF PubMed Google Scholar). A novel Src homologous adapter protein, SLAP, also binds ligand-activated EphA2 (14Pandey A. Duan H. Dixit V.M. J. Biol. Chem. 1995; 270: 19201-19204Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). EphA4 interacts with the Src family kinase, p59 fyn, through the major phosphorylation site at position Tyr602 (15Ellis C. Kasmi F. Ganju P. Walls E. Panayotou G. Reith A.D. Oncogene. 1996; 12: 1727-1736PubMed Google Scholar). We recently showed that ligand-activated EphB1 recruits two different adapter proteins, Grb2 and Grb10, through their respective SH2 domains (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Two distinct EphB1 subdomains are involved. The Grb10 SH2 domain binds EphB1 through Tyr929 (17Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar), a residue within the conserved, carboxyl-terminal sterile α motif that is shared among all Eph family receptors and a wide range of other signaling proteins (18Schultz J. Ponting C.P. Hofman K. Bork P. Protein Sci. 1997; 6: 249-253Crossref PubMed Scopus (270) Google Scholar). In contrast, Grb2 binds residues within the catalytic domain (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Here we used a yeast two-hybrid interaction screen to identify a third SH2 containing adapter protein, Nck (19Lehman J.R. Riethmuller G. Johnson J.P. Nucleic Acids Res. 1990; 18: 1048Crossref PubMed Scopus (160) Google Scholar), as one that interacts with EphB1 upon ligand activation. Unlike Grb10 and Grb2, the Nck SH2 domain binds EphB1 at a juxtamembrane tyrosine residue that is required for ligand activation of c-Jun kinase. Functional studies identify an important role for this residue in mediating cell attachment to fibronectin. Fusion plasmids were constructed to permit shuttling of EphB1-encoding inserts from the pAC-GST 1The abbreviations used are: GST, glutathioneS-transferase; HRMEC, human renal microvascular endothelial cells; HA, hemagglutinin; PCR, polymerase chain reaction; JNK, c-Jun kinase; GTPγS, guanosine 5′-3-O-(thio)triphosphate. (Pharmingen, San Diego, CA) expression vector to the yeast two-hybrid "bait" LexA fusion plasmid pBTM116 (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Parent sequences were derived from the predominant human EphB1 cDNA recovered from the human renal microvascular endothelial cell library (HRMEC) (HuELKI/hEphB1), and amino acid designations refer to GenBank™ AF037331. The eukaryotic expression construct, pSRα-hEphB1-HA, was constructed by appending sequences encoding a tandem repeated hemagglutinin (HA) epitope tag (20Jyers M. Tokiwa G. Nash R. Futcho B. EMBO J. 1992; 11: 1773-1784Crossref PubMed Scopus (339) Google Scholar) to the carboxyl terminus of the hEphB1 cDNA. 2E. Stein, A. A. Lane, D. P. Cerretti, H. O. Shoecklmann, A. D. Schroff, R. L. Van Etten, and T. O. Daniel, submitted for publication. Overlap extension PCR was used to generate the EphB1cy mutations, EphB1cyY594F and EphB1cyY600F (21Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). Oligonucleotide 1 (5′ primer, 5′-CTGGTTCCGGCGATCCCGGGGAGGAAACGGGCTTATAGC-3′, EphB1 sequence, underlined) and a specific 3′ primer encoding the mutation Y594F (primer Y594Frev, 5′-GGGGTCAATGAAGATCTTCATCCC-3′) or Y600F (primer Y600Frev, 5′-GGGATCCTCGAAAGTGAAGGGGTC-3′) were used to generate the 5′ PCR product; oligonucleotide 2 (3′ primer, 5′-CTCGCTCCGGCGAGGTCGACGTCATGCCATTGCCGTTGG-3′) and a specific 5′ primer encoding mutation Y594F (5′-ATGAAGATCTTCATTGACCCCTTC-3′) or Y600F (5′-CCCTTCACTTTCGAGGATCCCAAC-3′) were used to generate the 3′-overlapping PCR products. These products were reannealed, PCR amplified, and digested with BglII and Bsu36I. The recovered fragments were cloned intoBglII/Bsu36I digested pAC-GST/EphB1cyto generate pAC-GST/EphB1cyY594F and pAC-GST/EphB1cyY600F, respectively. Products were confirmed by sequence analysis, and the SmaI-SalI fragments of EphB1cyY594F and EphB1cyY600F were substituted for the SmaI-SalI fragment of EphB1cy pBTM116/EphB1cy. To generate pSRα-hEphB1-HA-Y594F and pSRα-hEphB1-HA-Y600F, the corresponding BglII-Bsu36I fragment was substituted for the BglII-Bsu36I fragment of pSRα-hEphB1-HA. Plasmids pBTM116-EphB1cyK652R and pACT/DOCK were described previously (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 22Clemens J.C. Ursuliak Z. Clemens K.K. Price J.V. Dixon J.E. J. Biol. Chem. 1996; 271: 17002-17005Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). COS 1 cells were passaged in Dulbecco's modified Eagle's growth medium containing 10% defined supplemented calf serum (HyClone Laboratories, Logan, UT). P19 cells were cultured in α-modified minimum essential medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. HRMEC were cultured as described (23Martin M.M. Schoecklmann H.O. Foster G. Barley-Maloney L. McKanna J. Daniel T.O. In Vitro Cell. Dev. Biol. 1997; 33: 261-269Crossref Scopus (13) Google Scholar). The genotype of theSaccharomyces cerevisiae reporter strain L40 is MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ(24Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). Growth medium and culture conditions used were as described (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar,25Rose M.S. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar). The yeast reporter strain L40, containing the reporter geneslacZ and HIS3 downstream of a LexA promotor, was sequentially transformed with the pLexA-EphB1cy and then with a cDNA library encoding VP16 (transcriptional activation domain) fusions with the peptide sequences expressed in murine embryos (embryonic days 9.5 and 10.5) using the lithium acetate method (26Hill J. Donald K.A. Griffins D.E. Nucleic Acids Res. 1991; 19: 5791Crossref PubMed Scopus (460) Google Scholar,27Schiestl R.H. Giest R.D. Curr. Genet. 1989; 16 (346): 399Google Scholar). Among a total of 2 × 107 yeast transformants, 320 His(+) colonies were isolated. Of 93 plasmids selected and sequenced for specific interaction with the pLexA-EphB1cybait, 6 independent plasmids encode the SH2 domain of Nck (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 19Lehman J.R. Riethmuller G. Johnson J.P. Nucleic Acids Res. 1990; 18: 1048Crossref PubMed Scopus (160) Google Scholar). Analysis of EphB1 subdomain deletions was conducted as described (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Cells were transfected using LipofectAMINE (Life Technologies, Inc.) as described by the manufacturer. About 40 h after transfection, the medium was removed, and cells were incubated for 5 h at 37 °C with Opti-MEM medium containing 0.5 mm sodium suramin (RBI, Natick, MA). Cells were then washed three times with serum-free medium, re-equilibrated in medium for 1 h, and then incubated at 37 °C for 10 min with agonists as described (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Cells were lysed in buffer D (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and 250 μg of the clarified cell lysate protein was incubated with the indicated antibodies for 6–12 h at 4 °C. Endogenous EphB1 was immunoprecipitated using rabbit anti-EphB1 (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) (see Fig.2 A), or exogenous, epitope-tagged EphB1 (see Fig.2 B) was immunoprecipitated using anti-hemagglutinin monoclonal, C12A5 (Boehringer Mannheim). Immunoprecipitates were recovered on protein A-Sepharose beads, washed extensively, and separated on 10% SDS-polyacrylamide gel electrophoresis gels under nonreducing conditions (omitting dithiothreitol from the loading buffer). Immunoblots were incubated with rabbit antiserum to the cytoplasmic domain of rat EphB1 (Santa Cruz Biotechnology, Santa Cruz, CA), murine monoclonal antibodies to Nck (Santa Cruz), or anti-phosphotyrosine 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY). P19 (see Fig. 3 A) or human HRMEC were serum-starved in Opti-MEM medium for 24–30 h before stimulation with unclustered ephrin B1/Fc (500 ng ml−1) or pre-clustered, multimeric ephrin B1/Fc (500 ng ml−1 ephrin B1/Fc + 50 ng ml−1 anti-Fc). Transfected P19 cells (see Fig.3 B) were serum-starved in Opti-MEM medium for 12–15 h. Cells were then replated on fibronectin-coated 60-mm dishes, allowed to attach for 120 min, and then incubated with agonist for the indicated times at 37 °C. Cells were lysed at 4 °C in RIPA buffer (50 mm Tris-Cl, pH 7.2, 150 mm NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 20 mmβ-glycerophosphate, 100 μmsodium-o-vanadate, 1 mm phenylmethylsulfonyl fluoride, 2 μg ml−1 aprotinin, 0.5 μg ml−1 leupeptin), and lysates were clarified by centrifugation. Endogenous JNK was immunoprecipitated with monoclonal anti-JNK antibody (Santa Cruz) as described (28Westwick J.K. Brenner D.A. Methods Enzymol. 1995; 255: 342-359Crossref PubMed Scopus (46) Google Scholar). GST-c-Jun (1–135) was used as a substrate in kinase reactions (29Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmand M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar). Reaction products were separated by SDS-polyacrylamide gel electrophoresis, transferred to Immobilin-P (Millipore), and subjected to autoradiography. Equal loading of GST-c-Jun (kinase substrate) was verified by Amido Black staining, and equal recovery of JNK antigen was confirmed by immunoblot using rabbit anti-JNK (Santa Cruz). Following autoradiography, stained substrate bands were subjected to either scintillation counting or PhosphorImager analysis. Six-well plates (Falcon) were coated with thin layers of fibronectin (0.5 μg cm−2) as described (30Ingber D.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3579-3583Crossref PubMed Scopus (409) Google Scholar). Growth medium was replaced 24 h before harvest with binding medium, α-minimum essential medium containing 1% bovine albumin. Transfected cells were recovered by brief trypsinization, washed three times with binding medium, and then plated at 1 × 105 cells/well. Preclustered ephrin B1/Fc (500 ng ml−1 ephrin B1/Fc + 50 ng ml−1 anti-Fc) was added coincident with plating. After 90 min, unattached cells were dislodged by applying four brisk slaps of the plate on a horizontal surface. The attached cell layer was carefully washed once with phosphate-buffered saline containing calcium and magnesium to collect the remaining unattached cells. Adherent cells were collected by incubation in Dispase as described (Collaborative Biomedical Products), recovered by centrifugation, and washed, and the viable cells were counted. The ratio of attached to total number of cells recovered was calculated for each of three wells. Data are expressed as the means ± S.E. and are representative of three independent experiments. To identify signaling molecules that interact with EphB1, we conducted a yeast two-hybrid screen of a murine embryonic day 9.5 and 10.5 cDNA library using the cytoplasmic domain of EphB1 (amino acids 556–985) (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Among 290 clones selected, 93 independent partial cDNAs were sequenced; of these, 6 included nonidentical overlapping cDNA fragments that encode the SH2 domain of Nck (19Lehman J.R. Riethmuller G. Johnson J.P. Nucleic Acids Res. 1990; 18: 1048Crossref PubMed Scopus (160) Google Scholar). The Nck domains recovered as independent clones are represented in Fig. 1 A. To evaluate the biochemical basis for the EphB1-Nck interaction, we generated a number of mutations in the EphB1cy bait. Shown in Fig. 1 B, an intact EphB1cy tyrosine kinase function was required for Nck binding. Mutation of the ATP-binding lysine (K652R) interrupted the two-hybrid interaction. This finding is consistent with role of SH2 domains in binding phosphotyrosine-containing peptides (31Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar). To further define the site at which Nck binds EphB1, we created a series of EphB1cy domain deletions, including removal of the juxtamembrane domain (pLexA-EphB1cyΔJM), the carboxyl-terminal domain (pLexA-EphB1cyΔCterm), or both (pLexA-EphB1cyΔJM/Cterm) (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In each case, the tyrosine kinase catalytic domain was retained to permit generation of an SH2 binding site through tyrosine self-phosphorylation. The juxtamembrane domain (amino acids 556–617) of EphB1cy was required for Nck interaction (Fig. 1 B). Based on published data showing that the Nck-SH2 domain preferentially binds tyrosine phosphopeptides with the sequence (Tyr(P)-hydrophilic-hydrophilic-Pro) (31Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar), we used site-directed mutagenesis to evaluate the two candidate tyrosine residues of EphB1cy within the juxtamembrane domain, Tyr594 and Tyr600. A single substitution, Y594F, disrupted the two-hybrid interaction between EphB1cyand the Nck SH2 domain. The second consensus site, EphB1cyY600, appears not to be required for the interaction (Fig. 1 B). Like Eph receptors, recent studies have implicated Nck in neural targeting. Disordered retinal neuron assembly was observed when the Drosophila Nck homologue, DOCK, was disrupted (32Garrity P. Rao Y. Salecker I. McGlade J. Pawson T. Zipursky S.L. Cell. 1996; 85: 639-650Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Shown in Fig. 1 C, a DOCK fusion (22Clemens J.C. Ursuliak Z. Clemens K.K. Price J.V. Dixon J.E. J. Biol. Chem. 1996; 271: 17002-17005Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) interacts with EphB1cy bait in the yeast two-hybrid system. Like Nck, the DOCK interaction requires both intact EphB1 tyrosine kinase function and Tyr594. To evaluate the significance of these yeast two-hybrid results, we tested whether Nck co-precipitates with EphB1 following ligand activation in cells that express endogenous EphB1, HRMEC (23Martin M.M. Schoecklmann H.O. Foster G. Barley-Maloney L. McKanna J. Daniel T.O. In Vitro Cell. Dev. Biol. 1997; 33: 261-269Crossref Scopus (13) Google Scholar), and P19 embryonal carcinoma cells (33Bain G. Ray W.J. Yao M. Gottlieb D.I. BioEssays. 1994; 15: 343-348Crossref Scopus (236) Google Scholar). EphB1 binds and is activated by ephrin B1/Fc (LERK-2/Fc) (34Beckmann M.P. Cerretti D.P. Baum P. Vanden Bos T. James L. Farrah T. Kozlosky C. Hollingsworth T. Shilling H. Maraskovsky E. Fletcher F.A. Lhotak V. Pawson T. Lyman S.D. EMBO J. 1994; 13: 3757-3762Crossref PubMed Scopus (151) Google Scholar,35Davis S. Gale N.W. Aldrich T.H. Maisonpierre P.C. Lhotak V. Pawson T. Goldfarb M. Yancopoulos G.D. Science. 1994; 226: 816-819Crossref Scopus (633) Google Scholar). Because previous reports have demonstrated differences in response to ephrin B1/Fc, depending upon whether it is presented as a dimer or as an anti-Fc clustered multimer (5Wang H.U. Anderson D.J. Neuron. 1997; 18: 383-396Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar),2 both were evaluated. As shown in Fig. 2 A, ephrin B1/Fc (dimeric and clustered multimer) stimulated tyrosine phosphorylation of EphB1. Nck was recovered in EphB1 complexes following ligand activation. Based on the yeast two-hybrid data presented above, we proceeded to evaluate the role of EphB1-Tyr594 in the Nck interaction. Shown in Fig. 2 B, we expressed HA epitope-tagged versions of either wild type or mutant (Y594F or Y600F) EphB1 in Cos-1 cells. Ephrin B1/Fc stimulated tyrosine phosphorylation of transiently expressed EphB1. Qualitatively similar EphB1 tyrosine phosphorylation was observed in EphB1 wild type and mutants (Y594F and Y600F), consistent with our data showing that a number of cytoplasmic domain tyrosine residues undergo phosphorylation upon ligand activation (16Stein E. Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; 271: 23588-23593Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Nck was recruited to wild type and the Y600F mutant EphB1 but not to Y594F mutant EphB1 receptors. In aggregate, these findings provide strong evidence that Nck recruitment requires phosphorylation of Tyr594. We anticipated that cytoskeletal rearrangements are a necessary feature for cell-cell aggregation and targeting functions subserved by Eph receptors. In addition, recent work showed that Nck binds a serine threonine kinase, NIK, that serves as an upstream regulator of the JNK signaling pathway (36Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (219) Google Scholar). Based on these observations, we evaluated the potential for Nck to couple EphB1 with JNK activation. Shown in Fig.3 A, c-Jun kinase was activated when P19 cells were exposed to ephrin B1/Fc. Similar results were obtained in HRMEC (not shown). As with the recruitment of Nck (Fig. 2), dimeric or multimeric ephrinB1/Fc evoked similar JNK activation responses. These effects were seen at ephrin B1/Fc concentrations greater than 125 ng ml−1, and activation was not stimulated by human IgG1, which is used as a control for Fc domain effects (not shown). JNK activity increases of 2–3-fold were typically seen within 10 min and in some experiments increased to 5-fold by 120 min (Fig. 3 A, right panel). This timing pattern is consistent with that of Nck recruitment to ligand-activated EphB1. Nck is found in EphB1 complexes as early as 7 min and persists beyond 30 min.2 A similarly delayed JNK activation response has been observed in response to transforming growth factor-β, where persistent increases in activity are evident between 2 and 12 h (37Atfi A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). To evaluate functional consequences of Nck recruitment upon EphB1-mediated responses, we used a dominant negative strategy to undermine the effects coupled to endogenous receptor activation. By expressing Nck binding-defective mutant EphB1 receptors at sufficiently high levels in P19 cells, we could evaluate changes in JNK activation and attachment to fibronectin-coated plates. Our transfection methods achieve high efficiency transfection of P19 cells (60–70%), and the pSRα expression plasmids drive exogenous EphB1 expression levels to 20–40-fold those of endogenous receptors. Using this approach, we assessed dominant effects of wild type, kinase defective mutant (K652R), or Nck binding mutant (Y594F) EphB1 upon downstream JNK activation and cell attachment. Shown in Fig. 3 B, ephrin B1 stimulated 2–2.5-fold increases in JNK activity in cells transfected with either vector control (pSRα) or wild type EphB1 (pSRα-EphB1/HA). In contrast, ephrin B1 failed to increase JNK activity in cells transfected with either kinase defective (K652R) or Nck binding defective (Y594F) mutant receptors. In multiple experiments, we consistently observed a lower activation of JNK in transfected (2–2.5-fold), compared with nontransfected cells (3–5-fold). This appears to reflect differences in basal JNK activation, depending upon the transfection protocol. Correlating with the JNK activation results, identically treated transfected P19 cells showed marked increases in attachment to fibronectin-coated dishes when transfected with either vector alone or wild type EphB1 expression plasmid (Fig. 3 B). Yet, high level expression of kinase defective (K652R) or Nck binding defective mutant (Y594F) EphB1 receptors eliminated ephrin B1-promoted attachment. In aggregate, our findings support a role for Nck in JNK activation and attachment responses downstream of EphB1 activation Nck is an EphB1-interactive protein that is recruited to EphB1 signaling complexes, apparently through binding of its SH2 domain with EphB1 residue Tyr594. This interaction is stimulated by ligand activation in P19 cells and HRMEC expressing native receptors. EphB1 receptor activation stimulates JNK, an effect requiring the recruitment of Nck to EphB1. Mutation of the site at which Nck binds EphB1 attenuates downstream JNK activation and EphB1-coupled attachment responses. Identification of Tyr594 as the site of interaction in the yeast two-hybrid system was somewhat surprising. Based on its affinity for small phosphopeptides, a consensus recognition binding site for the Nck SH2 domain, pYDEP, was determined (31Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar). This consensus is different from the residues adjacent to Tyr594 (YIDP) and Tyr600 (YEDP) in the EphB1 sequence. Both of these motifs are shared between EphB1 and EphB2 (15Ellis C. Kasmi F. Ganju P. Walls E. Panayotou G. Reith A.D. Oncogene. 1996; 12: 1727-1736PubMed Google Scholar). We have confirmed the role of Tyr594 in Nck binding in both yeast and mammalian cell systems through experiments that included independent review of the sequences of each construct shown in Fig. 2 B. A previous report identified a platelet-derived growth factor β receptor peptide sequence, pYVPL, as a Nck binding site (38Nishimura R. Kashishian A. Li W. Mondino A. Zhou M. Cooper J. Schlessinger J. Mol. Cell. Biol. 1993; 13: 6889-6896Crossref PubMed Scopus (155) Google Scholar), suggesting that some flexibility exists in the binding requirements. It is noteworthy that the sequence, 594pY(I/V)DP, is conserved at this position in all the Eph family receptors (15Ellis C. Kasmi F. Ganju P. Walls E. Panayotou G. Reith A.D. Oncogene. 1996; 12: 1727-1736PubMed Google Scholar). The previous finding that Nck does not bind EphA4 (15Ellis C. Kasmi F. Ganju P. Walls E. Panayotou G. Reith A.D. Oncogene. 1996; 12: 1727-1736PubMed Google Scholar) suggests that factors other than primary amino acid sequence are likely determinants of the site of tyrosine phosphorylation in this subdomain. A recent report by Holland et al. provided evidence for an indirect role of Nck in signaling downstream of activated EphB2 (39Holland S.J. Gale N.W. Gish G.D. Roth R.A. Songyang Z. Cantley L.C. Yancopoulos G.D. Pawson T. EMBO J. 1997; 16: 3877-3888Crossref PubMed Scopus (240) Google Scholar). They found that EphB2 activation caused tyrosine phosphorylation of a 62–64-kDa protein (p62dok) (40Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; 88: 197-204Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 41Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), which in turn formed a complex with the Ras GTPase activation protein (RasGAP) and Nck. We have re-evaluated our EphB1 immunoprecipitates following ligand activation and were unable to demonstrate co-precipitation of either p62-dok or RAS-GAP with EphB1 recovered from P19 (data not shown). Despite the structural similarities of the EphB subclass receptor cytoplasmic domains, it appears that remarkable differences may be observed in the signaling pathways utilized by specific EphB receptors. The identification of Nck as an EphB1-interactive protein is particularly intriguing in light of the roles EphB subclass receptors play in neuronal guidance, targeting, and cell-cell aggregation (4Bohme B. VandenBos T. Cerretti D.P. Park L.S. Holtrich U. Ruebsamen-Waigmann H. Strebhardt K. J. Biol. Chem. 1996; 271: 24747-24752Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 5Wang H.U. Anderson D.J. Neuron. 1997; 18: 383-396Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar,42Henkemeyer M. Orioli D. Henderson J.T. Saxton T.M. Roder S. Pawson T. Klein R. Cell. 1996; 86: 35-46Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). The Nck-related Drosophila protein, DOCK, mediates signals that direct axonal guidance in the Drosophila eye, where DOCK mutations cause abnormal fasciculation of retinal axons, with failure to follow guidance cues to their correct targets (32Garrity P. Rao Y. Salecker I. McGlade J. Pawson T. Zipursky S.L. Cell. 1996; 85: 639-650Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Our findings show that DOCK displays functional similarities to Nck in its capacity to bind ligand-activated EphB1 and suggest that an upstream EphB receptor participates in the retinal axon targeting that is aberrant in the DOCK mutants. In other studies, Nck has been shown to interact with a number of crucial determinants of cytoskeletal function and signaling. Yeast two-hybrid screens have identified Nck interactions with the Wiskott Aldrich syndrome protein (WASP, a putative effector of CDC42) (43Rivero-Lezcano O.M. Marcilla A. Sameshima J.H. Robbins K.C. Mol. Cell. Biol. 1995; 15: 5725-5731Crossref PubMed Scopus (280) Google Scholar), and a serine/threonine protein kinase PRK2, similar to the Rho effector, PKN) (44Quilliam L.A. Lambert Q.T. Mickelson-Young L.A. Westwick J.K. Sparks A.B. Kay B.K. Jenkins N.A. Gilbert D.J. Copeland N.G. Channing J.D. J. Biol. Chem. 1996; 271: 28772-28776Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Nck associates with focal adhesion kinase upon stimulation of mesangial cells with thrombin, correlating with thrombin-stimulated focal adhesion kinase activation (45Choudhury G.G. Marra F. Abbound H.E. Am. J. Physiol. 1996; 270: F295-F300PubMed Google Scholar). Two independent observations may place Nck in responses mediated through Rac, the G-protein implicated in formation of lamellipodia required for migration (36Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (219) Google Scholar, 46Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). Nck SH3 domains bind with high affinity to mPak3, a Ste20 family serine/threonine kinase that binds Cdc42 and Rac1, but not RhoA, in their activated (GTPγS bound) states (46Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). As mentioned above, Nck also interacts with the Ste20 family serine/threonine kinase, NIK, a protein coupled to activation of MKK4 and JNK (36Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (219) Google Scholar). Other intermediary kinases may participate in JNK activation. For example, transforming growth factor-β stimulates delayed JNK activation through TAK1, a kinase capable of phosphorylating MKK4, a mediator of JNK activation (47Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Tangiguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). We demonstrate that Nck recruitment to EphB1, with the subsequent activation of JNK, appears necessary to link Eph receptor activation with cellular cytoskeletal modifications important in attachment. However, other recent results from our lab have shown an important role for ephrin B1 clustering in promoting P19 cell attachment.2As was shown here, Nck recruitment to EphB1 complexes does not require presentation of ephrin B1 as a preclustered multimer. Thus, Nck recruitment appears necessary but not sufficient for the functional coupling of EphB1 activation to this cell attachment response. We anticipate that Nck is one of several proteins that must function in this regard. We offer special thanks to Stan Hollenberg (Fred Hutchinson Cancer Research Institute, Seattle, WA) for the two-hybrid vectors and murine cDNA library, to James Clemens (Department of Biochemistry, University of Michigan, Ann Arbor, MI) for the DOCK construct, to John Kyriakis (Massachusetts General Hospital, Boston, MA) for the GST-c-Jun expression plasmid, and to Tony Pawson (Samuel Lunenfeld Research Institute, Toronto, Ontario) for helpful critical input.
Abstract Background N -3-oxo-tetradecanoyl- L -homoserine lactone (oxo-C14-HSL) is one of the N -acyl homoserine lactones (AHL) that mediate quorum sensing in Gram-negative bacteria. In addition to bacterial communication, AHL are involved in interactions with eukaryotes. Short-chain AHL are easily taken up by plants and transported over long distances. They promote root elongation and growth. Plants typically do not uptake hydrophobic long sidechain AHL such as oxo-C14-HSL, although they prime plants for enhanced resistance to biotic and abiotic stress. Many studies have focused on priming effects of oxo-C14-HSL for enhanced plant resistance to stress. However, specific plant factors mediating oxo-C14-HSL responses in plants remain unexplored. Here, we identify the Arabidopsis protein ALI1 as a mediator of oxo-C14-HSL-induced priming in plants. Results We compared oxo-C14-HSL-induced priming between wild-type Arabidopsis Col-0 and an oxo-C14-HSL insensitive mutant ali1 . The function of the candidate protein ALI1 was assessed through biochemical, genetic, and physiological approaches to investigate if the loss of the ALI1 gene resulted in subsequent loss of AHL priming. Through different assays, including MAP kinase activity assay, gene expression and transcriptome analysis, and pathogenicity assays, we revealed a loss of AHL priming in ali1 . This phenomenon was reverted by the reintroduction of ALI1 into ali1 . We also investigated the interaction between ALI1 protein and oxo-C14-HSL using biochemical and biophysical assays. Although biophysical assays did not reveal an interaction between oxo-C14-HSL and ALI1, a pull-down assay and an indirect method employing biosensor E. coli LuxCDABE support such interaction. We expressed fluorescently tagged ALI1 in tobacco leaves to assess the localization of ALI1 and demonstrate that ALI1 colocalizes with the plasma membrane, tonoplast, and endoplasmic reticulum. Conclusions These results suggest that the candidate protein ALI1 is indispensable for oxo-C14-HSL-dependent priming for enhanced resistance in Arabidopsis and that the ALI1 protein may interact with oxo-C14-HSL. Furthermore, ALI1 protein is localized in the cell periphery. Our findings advance the understanding of interactions between plants and bacteria and provide an avenue to explore desired outcomes such as enhanced stress resistance, which is useful for sustainable crop protection.
RNA-based disease control has shown great potential for controlling pest and diseases in crop plants. While delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants, which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Focusing on agronomic important barley - Fusarium spec. pathosystems, we have sought for alternative strategies to apply dsRNAs for fungal control. Recently, we have demonstrated that a spray application of a long noncoding dsRNA termed CYP3RNA, which targets the three fungal Cytochrome P450 lanosterol C-14α-demethylase genes FgCYP51A, FgCYP51B, and FgCYP51C, inhibits Fusarium graminearum (Fg) on barley leaves (Koch et al., PLoS Pathogens, 12, e1005901, 2016). Here we show that another Fusarium species, F. culmorum (Fc), also is sensitive to CYP51-derived dsRNAs. Treating Fc with various dsRNAs targeting the genes FcCYP51A, FcCYP51B and FcCYP51C was destructive to the fungus and resulted in growth retardation in in vitro cultures. We discuss important consequences of this finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential for novel plant protection strategies.
The ability of plants to monitor their surroundings, for instance the perception of bacteria, is of crucial importance. The perception of microorganism-derived molecules and their effector proteins is the best understood of these monitoring processes. In addition, plants perceive bacterial quorum sensing (QS) molecules used for cell-to-cell communication between bacteria. Here, we propose a mechanism for how N-acyl-homoserine lactones (AHLs), a group of QS molecules, influence host defense and fortify resistance in Arabidopsis thaliana against bacterial pathogens. N-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) primed plants for enhanced callose deposition, accumulation of phenolic compounds, and lignification of cell walls. Moreover, increased levels of oxylipins and salicylic acid favored closure of stomata in response to Pseudomonas syringae infection. The AHL-induced resistance seems to differ from the systemic acquired and the induced systemic resistances, providing new insight into inter-kingdom communication. Consistent with the observation that short-chain AHLs, unlike oxo-C14-HSL, promote plant growth, treatments with C6-HSL, oxo-C10-HSL, or oxo-C14-HSL resulted in different transcriptional profiles in Arabidopsis. Understanding the priming induced by bacterial QS molecules augments our knowledge of plant reactions to bacteria and suggests strategies for using beneficial bacteria in plant protection.
Netrins stimulate and orient axon growth through a mechanism requiring receptors of the DCC family. It has been unclear, however, whether DCC proteins are involved directly in signaling or are mere accessory proteins in a receptor complex. Further, although netrins bind cells expressing DCC, direct binding to DCC has not been demonstrated. Here we show that netrin-1 binds DCC and that the DCC cytoplasmic domain fused to a heterologous receptor ectodomain can mediate guidance through a mechanism involving derepression of cytoplasmic domain multimerization. Activation of the adenosine A2B receptor, proposed to contribute to netrin effects on axons, is not required for rat commissural axon outgrowth or Xenopus spinal axon attraction to netrin-1. Thus, DCC plays a central role in netrin signaling of axon growth and guidance independent of A2B receptor activation.
