Nck Recruitment to Eph Receptor, EphB1/ELK, Couples Ligand Activation to c-Jun Kinase*

Elke Stein, Uyen Huynh-Do, Andrew A. Lane, Douglas P. CerrettiDagger , and Thomas O. Daniel§

From the Departments of Pharmacology, Cell Biology, and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and Dagger  Immunex Corporation, Seattle, Washington 98101

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 its Drosophila 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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Eph family receptor tyrosine kinases transmit signals that direct cell migration, cell targeting, and cell-cell aggregation (1-5). 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 (6, 7).

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 (2, 8-10). For example, tectal gradients of the EphA3 (Mek4) ligand, ephrin A2 (ELF-1), direct developmental targeting of retinal axons expressing EphA3 receptor (11, 12). 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 (5). In a reconstituted system, 32D cells transfected with EphB3 (Hek2) receptors aggregate with cells transfected with the EphB3 ligand, ephrin B1 (4).

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 (13). A novel Src homologous adapter protein, SLAP, also binds ligand-activated EphA2 (14). EphA4 interacts with the Src family kinase, p59fyn, through the major phosphorylation site at position Tyr602 (15).

We recently showed that ligand-activated EphB1 recruits two different adapter proteins, Grb2 and Grb10, through their respective SH2 domains (16). Two distinct EphB1 subdomains are involved. The Grb10 SH2 domain binds EphB1 through Tyr929 (17), a residue within the conserved, carboxyl-terminal sterile alpha  motif that is shared among all Eph family receptors and a wide range of other signaling proteins (18). In contrast, Grb2 binds residues within the catalytic domain (16).

Here we used a yeast two-hybrid interaction screen to identify a third SH2 containing adapter protein, Nck (19), 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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of Recombinant Fusion Proteins, Baits, and Expression Constructs-- Fusion plasmids were constructed to permit shuttling of EphB1-encoding inserts from the pAC-GST1 (Pharmingen, San Diego, CA) expression vector to the yeast two-hybrid "bait" LexA fusion plasmid pBTM116 (16). 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 GenBankTM AF037331. The eukaryotic expression construct, pSRalpha -hEphB1-HA, was constructed by appending sequences encoding a tandem repeated hemagglutinin (HA) epitope tag (20) to the carboxyl terminus of the hEphB1 cDNA.2

Site-directed Mutagensis-- Overlap extension PCR was used to generate the EphB1cy mutations, EphB1cyY594F and EphB1cyY600F (21). 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 into BglII/Bsu36I digested pAC-GST/EphB1cy to 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 pSRalpha -hEphB1-HA-Y594F and pSRalpha -hEphB1-HA-Y600F, the corresponding BglII-Bsu36I fragment was substituted for the BglII-Bsu36I fragment of pSRalpha -hEphB1-HA. Plasmids pBTM116-EphB1cyK652R and pACT/DOCK were described previously (16, 22).

Cell Culture-- 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 alpha -modified minimum essential medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. HRMEC were cultured as described (23).

Yeast Two-hybrid Screen-- The genotype of the Saccharomyces cerevisiae reporter strain L40 is MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ (24). Growth medium and culture conditions used were as described (16, 25). The yeast reporter strain L40, containing the reporter genes lacZ 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 (26, 27). 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-EphB1cy bait, 6 independent plasmids encode the SH2 domain of Nck (16, 19). Analysis of EphB1 subdomain deletions was conducted as described (16).

Cell Transfection and Immunoprecipitation of Ligand-activated EphB1 with Nck-- 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 (16). Cells were lysed in buffer D (16), 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 (16) (see Fig. 2A), or exogenous, epitope-tagged EphB1 (see Fig. 2B) 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).

JNK Assays-- P19 (see Fig. 3A) 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. 3B) 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 mM beta -glycerophosphate, 100 µM sodium-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 (28). GST-c-Jun (1-135) was used as a substrate in kinase reactions (29). 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.

Cell Attachment Assays-- Six-well plates (Falcon) were coated with thin layers of fibronectin (0.5 µg cm-2) as described (30). Growth medium was replaced 24 h before harvest with binding medium, alpha -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EphB1 Interaction with Nck Requires an Active Tyrosine Kinase and Tyr594-- 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) (16). 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 (19). The Nck domains recovered as independent clones are represented in Fig. 1A.


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Fig. 1.  

Two-hybrid analysis for Nck. A, EphB1-interactive Nck cDNA clones overlap the Nck SH2 domain. Sequence overlap regions are depicted. The asterisk indicates the Nck prey used for all subsequent analyses. B, a tyrosine kinase active form of EphB1cy and tyrosine phosphorylation of EphB1cyY594 is required for Nck interaction. The reporter strain L40 was cotransformed with the indicated plasmids plated on medium lacking lysine, leucine, uracil, and tryptophan and grown for 3 days at 30 °C. Growth of the same yeast cotransfectants was compared on medium including or lacking histidine (not shown). Growth was assessed after 3 days at 30 °C. White bars, juxtamembrane domain; hatched bars, carboxyl-terminal domain; black bars, catalytic domain; *, site-directed mutant; +, growth on His-deficient plates; -, no growth. Included are two previously described EIPs, Grb2 and Grb10 (16), and c-Fyn, shown to interact with Tyr(P)600 in EphB1 and Tyr(P)602 in EphA4 (15). C, the Nck-related Drosophila protein, DOCK, interacts with EphB1. The reporter strain L40 was cotransformed with the indicated plasmids, and plated as described for B. Growth of the same yeast cotransfectants was compared on medium lacking lysine, leucine, uracil, and tryptophan including (-KLUT) or lacking histidine (-KLUTH). Growth was assessed and pictures were taken after 3 days at 30 °C. The DOCK interaction require a tyrosine kinase active form of EphB1cy and tyrosine phosphorylation of EphB1cyY594F.

To evaluate the biochemical basis for the EphB1-Nck interaction, we generated a number of mutations in the EphB1cy bait. Shown in Fig. 1B, 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 (31).

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-EphB1cyDelta JM), the carboxyl-terminal domain (pLexA-EphB1cyDelta Cterm), or both (pLexA-EphB1cyDelta JM/Cterm) (16). 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. 1B). Based on published data showing that the Nck-SH2 domain preferentially binds tyrosine phosphopeptides with the sequence (Tyr(P)-hydrophilic-hydrophilic-Pro) (31), 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 EphB1cy and the Nck SH2 domain. The second consensus site, EphB1cyY600, appears not to be required for the interaction (Fig. 1B).

Nck-related Drosophila Protein, DOCK, Interacts with EphB1cy-- 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 (32). Shown in Fig. 1C, a DOCK fusion (22) interacts with EphB1cy bait in the yeast two-hybrid system. Like Nck, the DOCK interaction requires both intact EphB1 tyrosine kinase function and Tyr594.

Ligand-activated EphB1 Recruits Nck-- 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 (23), and P19 embryonal carcinoma cells (33). EphB1 binds and is activated by ephrin B1/Fc (LERK-2/Fc) (34, 35). 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 (5),2 both were evaluated. As shown in Fig. 2A, ephrin B1/Fc (dimeric and clustered multimer) stimulated tyrosine phosphorylation of EphB1. Nck was recovered in EphB1 complexes following ligand activation.


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Fig. 2.   Phosphorylation of the human EphB1 Tyr594 is required for association with Nck. A, clustered and unclustered ephrin B1/Fc stimulates EphB1 tyrosine phosphorylation and coimmunoprecipitation of Nck. P19 cells were treated as indicated under "Materials and Methods," exposed for 10 min at 37 °C to the indicated ligand, either preclustered (+ anti-Fc) or unclustered (- anti-Fc) or no addition (NA), lysed, and then subjected to immunoprecipitation with EphB1 antibodies as described (16). B, Cos-1 cells were transfected with the indicated expression constructs ("Materials and Methods"). 40 h after transfection, cells were treated as described under "Materials and Methods," stimulated, and lysed as described above. Transfected EphB1 mutant (Y594F or Y600F) or wild type receptors were immunoprecipitated with anti-HA antibodies. Western blots were analyzed with either rabbit polyclonal anti-EphB1 (Santa Cruz), mouse monoclonal anti-phosphotyrosine, 4G10 (Upstate Biotechnology Inc.), or anti-Nck (Santa Cruz) antiserum as indicated.

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. 2B, 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 (16). 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.

Ephrin B1/Fc Activates c-Jun Kinase (JNK) through EphB1-Nck Interaction-- 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 (36). Based on these observations, we evaluated the potential for Nck to couple EphB1 with JNK activation. Shown in Fig. 3A, 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. 3A, 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-beta , where persistent increases in activity are evident between 2 and 12 h (37).


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Fig. 3.   Activation of JNK by ephrin B1/Fc. A, time course of JNK activation by ephrin B1/Fc. P19 cells (left panel) were treated as indicated under "Materials and Methods" and exposed to clustered and unclustered ephrin B1/Fc (500 ng ml-1) for the indicated times. Cell lysates (150 µg) were immunoprecipitated with a phosphospecific anti-JNK (Santa Cruz), and immunoprecipitates were subjected to in vitro kinase assay using GST-c-Jun (1-135) as substrate. The phosphorylated proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore), and visualized by autoradiography. From the same experiment, following autoradiography, the stained substrate bands were subjected to scintillation counting. Data are representative of five independent experiments. B, the Nck binding mutant EphB1(Y594F) is a dominant negative inhibitor of ephrin B1/Fc JNK activation and attachment. P19 cells were transfected with pSRa (vector), pSRalpha -hEphB1-HA (wt), pSRalpha -hEphB1-HA(K652R), or pSRalpha -hEphB1-HA(Y594F) as indicated using the LipofectAMINE (Life Technologies, Inc.) method. 40 h after transfection, cells were treated as described under "Materials and Methods," stimulated for 120 min, lysed, immunoprecipitated, and assayed for JNK kinase activity as described above. Cells from the same transfection were assayed for attachment to fibronectin-coated plates as described under "Materials and Methods."

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 pSRalpha 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. 3B, ephrin B1 stimulated 2-2.5-fold increases in JNK activity in cells transfected with either vector control (pSRalpha ) or wild type EphB1 (pSRalpha -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. 3B). 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

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (31). 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 (15). 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. 2B. A previous report identified a platelet-derived growth factor beta  receptor peptide sequence, pYVPL, as a Nck binding site (38), 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 (15). The previous finding that Nck does not bind EphA4 (15) 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 (39). They found that EphB2 activation caused tyrosine phosphorylation of a 62-64-kDa protein (p62dok) (40, 41), 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 (4, 5, 42). 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 (32). 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) (43), and a serine/threonine protein kinase PRK2, similar to the Rho effector, PKN) (44). Nck associates with focal adhesion kinase upon stimulation of mesangial cells with thrombin, correlating with thrombin-stimulated focal adhesion kinase activation (45).

Two independent observations may place Nck in responses mediated through Rac, the G-protein implicated in formation of lamellipodia required for migration (36, 46). 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 (GTPgamma S bound) states (46). As mentioned above, Nck also interacts with the Ste20 family serine/threonine kinase, NIK, a protein coupled to activation of MKK4 and JNK (36). Other intermediary kinases may participate in JNK activation. For example, transforming growth factor-beta stimulates delayed JNK activation through TAK1, a kinase capable of phosphorylating MKK4, a mediator of JNK activation (47).

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.2 As 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by Public Health Service Awards DK38517 and DK47078 (to T. O. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: MCN S3223, Vanderbilt University Medical Center, 21st & Garland St., Nashville, TN 37232. Tel.: 615-343-8496; Fax: 615-343-7156; E-mail: tom.daniel{at}mcmail.vanderbilt.edu.

1 The abbreviations used are: GST, glutathione S-transferase; HRMEC, human renal microvascular endothelial cells; HA, hemagglutinin; PCR, polymerase chain reaction; JNK, c-Jun kinase; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

2 E. Stein, A. A. Lane, D. P. Cerretti, H. O. Shoecklmann, A. D. Schroff, R. L. Van Etten, and T. O. Daniel, submitted for publication.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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