From the Departments of Pharmacology, Cell Biology, and Medicine,
Vanderbilt University School of Medicine, Nashville, Tennessee
37232 and Immunex Corporation,
Seattle, Washington 98101
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ABSTRACT |
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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.
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INTRODUCTION |
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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 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.
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MATERIALS AND METHODS |
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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, pSR-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 pSR
-hEphB1-HA-Y594F and
pSR
-hEphB1-HA-Y600F, the corresponding BglII-Bsu36I
fragment was substituted for the BglII-Bsu36I fragment of
pSR
-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 -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 ml1) 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
-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 cm2) as
described (30). Growth medium was replaced 24 h before harvest
with binding medium,
-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.
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RESULTS |
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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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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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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 ml1, 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-
, where persistent increases in activity are evident
between 2 and 12 h (37).
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DISCUSSION |
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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 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 (GTPS 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-
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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; GTPS, 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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REFERENCES |
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