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Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry

Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry
Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry
Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling.
animal proteomics, bn-page, ms, protein–protein interactions, signal transduction
1615-9853
4514-4528
Darie, Costel C.
d5a4c384-25cc-4211-b991-b4fa8ec5c88b
Deinhardt, Katrin
5f4fe23b-2317-499f-ba6d-e639a4885dc1
Zhang, Guoan
fbb6a65f-dba6-43b8-aa9b-9a95f8c8a979
Cardasis, Helene S.
513ea464-fd33-473e-91c2-d0459da6ba8a
Chao, Moses V.
823c00e2-9f32-4a7b-a2c4-76e7c1fbf83d
Neubert, Thomas A.
bd2d3132-b85d-49fa-b879-488ac0e32ec7
Darie, Costel C.
d5a4c384-25cc-4211-b991-b4fa8ec5c88b
Deinhardt, Katrin
5f4fe23b-2317-499f-ba6d-e639a4885dc1
Zhang, Guoan
fbb6a65f-dba6-43b8-aa9b-9a95f8c8a979
Cardasis, Helene S.
513ea464-fd33-473e-91c2-d0459da6ba8a
Chao, Moses V.
823c00e2-9f32-4a7b-a2c4-76e7c1fbf83d
Neubert, Thomas A.
bd2d3132-b85d-49fa-b879-488ac0e32ec7

Darie, Costel C., Deinhardt, Katrin, Zhang, Guoan, Cardasis, Helene S., Chao, Moses V. and Neubert, Thomas A. (2011) Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry. Proteomics, 11 (23), 4514-4528. (doi:10.1002/pmic.201000819). (PMID:21932443)

Record type: Article

Abstract

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling.

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More information

e-pub ahead of print date: 28 October 2011
Published date: December 2011
Keywords: animal proteomics, bn-page, ms, protein–protein interactions, signal transduction
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 349688
URI: http://eprints.soton.ac.uk/id/eprint/349688
ISSN: 1615-9853
PURE UUID: d857357f-0149-4140-8ce1-5e20c4267194
ORCID for Katrin Deinhardt: ORCID iD orcid.org/0000-0002-6473-5298

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Date deposited: 11 Mar 2013 11:25
Last modified: 29 Oct 2019 01:39

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