Nck Recruitment to Eph Receptor, EphB1/ELK, Couples Ligand Activation to c-Jun Kinase (original) (raw)

1998, Journal of Biological Chemistry

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, Tyr 594 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 (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 pro