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 ß-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.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Chimerin Proteins/metabolism , Oncogene Proteins/metabolism , Proto-Oncogene Proteins/metabolism , RNA-Binding Proteins/metabolism , Receptor, EphB2/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Cell Separation , Chimerin Proteins/chemistry , Ephrin-B1/genetics , Ephrin-B1/metabolism , HEK293 Cells , Humans , Mass Spectrometry , Oncogene Proteins/chemistry , Phosphorylation , Protein Binding , Protein Subunits/chemistry , Protein Subunits/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , RNA Interference , RNA, Small Interfering/metabolism , RNA-Binding Proteins/chemistry , Receptor, EphB2/chemistry , Receptor, EphB2/genetics , Signal Transduction , src Homology DomainsABSTRACT
Glycosylated proteins on the cell surface have been shown to be essential for cell-cell interactions in development and differentiation. Our ultimate goal is to identify Asn-linked oligosaccharides that are directly involved in these critical in vivo functions. Because such oligosaccharides would be expected to reside on the integral plasma membrane proteins, and conventional two-dimensional gel techniques are ineffective at separating such proteins, we have developed a new approach to their identification on a proteomics scale from Caenorhabditis elegans. Membrane proteins are solubilized in guanidine-HCl, precipitated, and digested with trypsin. The glycopeptides are then separated by lectin chromatography. Next, glycopeptidase F digestion removes the oligosaccharides from the peptides and converts to Asp each Asn to which one was attached. The peptides are then analyzed by matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) mass spectrometry. Thus, the membrane glycoproteins are identified through the sequence tags of these peptides and the conversion of at least one deduced Asn residue to Asp at the Asn-X-Ser/Thr consensus sequence. To validate the utility of this approach, we have identified 13 membrane-bound N-glycosylated proteins from the major peaks observed on MALDI-Q-TOF analysis of our total glycopeptide fraction.
Subject(s)
Asparagine/metabolism , Membrane Proteins/isolation & purification , Oligosaccharides/metabolism , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Glycoproteins/isolation & purification , Glycoproteins/metabolism , Membrane Proteins/metabolism , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Glycosylation is one of the most common post-translational protein modifications. Carbohydrate-mediated interactions between cells and their environment are important in differentiation, embryogenesis, inflammation, cancer and metastasis and other processes. Humans and mice with mutations that prevent normal N-glycosylation show multi-systemic defects in embryogenesis, thereby proving that these molecules are essential for normal development; however, a large number of proteins undergo defective glycosylation in these human and mouse mutants, and it is therefore difficult to determine the precise molecular roles of specific N-glycans on individual proteins. We describe here a 'functional post-translational proteomics' approach that is designed to determine the role of N-glycans on individual glycoproteins in the development of Caenorhabditis elegans.
