Abstract
Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and β-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.
Highlights
Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation
We previously identified SH2 domain– containing adaptor protein B (Shb) as a potential signaling protein required for Eph- and ephrin-mediated in vitro cell sorting in a large-scale siRNA screen in HEK293 cells [13]
Compared with the negative control, where EphB2ϩ cells were co-incubated with parental HEK293 (Fig. 1A), incubation with ephrinB1-expressing cells forced the EphB2ϩ cells into compact clusters with cell crowding toward
Summary
The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2. Soliman§, Claus Jørgensen¶, Monika Tucholska§, and Robert Rottapel‡ʈ**2 From the ‡Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario M5S 1A8, Canada, the §LunenfeldTanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, the ¶Cancer Research UK Manchester Institute, University of Manchester, Alderley Park SK10 4TG, United Kingdom, the ʈDepartments of Medicine, Immunology and Medical Biophysics, University of Toronto, Toronto, Ontario M5S, Canada, and the **Division of Rheumatology, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada
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