The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2

• Published in: The Journal of biological chemistry 2020-03, Vol.295 (12), p.3932-3944
• Main Authors: Wagner, Melany J., Hsiung, Marilyn S., Gish, Gerald D., Bagshaw, Rick D., Doodnauth, Sasha A., Soliman, Mohamed A., Jørgensen, Claus, Tucholska, Monika, Rottapel, Robert
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - antagonists & inhibitors; Adaptor Proteins, Signal Transducing - chemistry; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Cell Separation; Chimaerin; Chimerin Proteins - chemistry; Chimerin Proteins - metabolism; EphB2, Eph receptor tyrosine kinase; Ephrin-B1 - genetics; Ephrin-B1 - metabolism; GTPase-activating protein (GAP); HEK293 Cells; Humans; Mass Spectrometry; Nck; Oncogene Proteins - chemistry; Oncogene Proteins - metabolism; p120 RasGAP; Phosphorylation; phosphotyrosine signaling; Protein Binding; Protein Subunits - chemistry; Protein Subunits - metabolism; Proto-Oncogene Proteins - antagonists & inhibitors; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; receptor tyrosine kinase; Receptor, EphB2 - chemistry; Receptor, EphB2 - genetics; Receptor, EphB2 - metabolism; RNA Interference; RNA, Small Interfering - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Shb; Signal Transduction; Src homology 2 domain (SH2 domain); src Homology Domains
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.009276

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.