Nck Recruitment to Eph Receptor, EphB1/ELK, Couples Ligand Activation to c-Jun Kinase*
- From the Departments of Pharmacology, Cell Biology, and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and ‡Immunex Corporation, Seattle, Washington 98101
Abstract
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.
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.
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↵§ 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.
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↵1 The 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.
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↵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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- Received November 19, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
