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

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.

Figure 1

A workflow for the biochemical and MS characterization of ephrin signaling. Unstimulated (−) or stimulated (+) cells were lysed and the lysates (and/or the pY99-IPs from the lysate) were separated by BN-PAGE. The protein complexes were characterized by Western blotting and/or by MS, followed by comparison with the current literature.

Figure 2

Analysis of lysates of (−) and (+) cells by SDS-PAGE and BN-PAGE. The lysates of (−) and (+) cells were separated by SDS-PAGE (A) and BN-PAGE 1D (B) and blotted to membranes and analyzed by WB using pY99 antibodies. The gel lanes from BN-PAGE 1D were excised and further reduced and denatured and separated in 2D SDS-PAGE and electroblotted by WB and analyzed with pY99 antibodies (C). In (C), the lysates were from (−) cells (0′) or cells stimulated with ephrinB1-Fc for 5, 10, 15, 20 and 45 minutes. The direction of migration in BN-PAGE is indicated by arrows followed by either 1D or 2D. The mass markers are indicated for each gel in 1D or 2D.

Figure 3

Representation of the proteins found by BN-PAGE and MS of the pY-IPs of (−) and (+) cells. A: Identification of well known protein interactions by analysis of small scale pY-IP in the presence of NP-40 detergent. The pY-IP from (−) and (+) cells were separated by BN-PAGE and the gel lanes were excised, cut into pieces/bands, digested by trypsin, and analyzed by MS. The approximate mass of the bands of interest was between 110–130 and 250–300 kDa. The proteins identified are labeled with different colors, shown at the bottom of the figure. B: as in A, except that identification of well known protein interactions were performed by analysis of larger scale pY-IP in the presence of DDM detergent. The mass markers are indicated for each gel.

Figure 4

Representation of selected proteins found by BN-PAGE and MS of the larger scale pY IPs of (−) and (+) cells. A: Proteins found by BN-PAGE and MS in either (−) or (+) cells or in both and shifted their molecular mass from low to high or from high to lo w. B: Proteins found by BN-PAGE and MS in both (−) and (+) cells that did not change in measured molecular mass (did not shift in BN-PAGE) upon ephrinB1-Fc stimulation, but changed their abundance by either increase or decrease of their relative amounts in the pY IP experiment.

Figure 5

Comparison of the intensities of MS spectra for peaks corresponding to peptides that are part of proteins found by BN-PAGE and MS. A: The peak with m/z of 906.52 (2+) that corresponds to a Trk-fused gene peptide QSTQVMAASMSAFDPLK has a higher intensity of this peptide in the pY-IP of (+) cells, compared with the (−) cells. Total ion count over one minute retention time was 4.46e3 versus 1.01e3. B: The peak with m/z of 695.71 (3+) that corresponds to a Calnexin peptide IADPDAVKPDDWDEDAPSK, has a lower intensity of this peptide in the pY-IP of (−) cells, compared with the (+) cells. Total ion count over one minute retention time was 1.25e3 versus 6.04e3. C: The peak with m/z of 1107.90 (2+) that corresponds to an Actin peptide DLYANTVLSGGTTMYPGIADR, had almost no change in the intensities of this peptide is observed in the pY-IP of both (−) and (+) cells. Total ion count over one minute retention time was 9.40e3 versus 8.62e3. The intensity scale for the spectra from both (−) and (+) cells for each individual peptide was identical.

Figure 6

Analysis of (−) and (+) cells by BN-PAGE and WB. The lysates or pY-IPs were separated by BN-PAGE 1D and SDS-PAGE 2D and then analyzed by WB with different antibodies. A: Analysis of lysates and pY-IPs of both (−) and (+) cells by BN-PAGE and WB using EphB2 antibodies. B: Analysis of lysates of (−) and (+) cells by BN-PAGE and WB using FAK and p130Cas antibodies. C: Analysis of lysates of (−) and (+) cells by BN-PAGE and WB using Nischarin, HRS, Nck2, EphB3, GAPDH, and actin antibodies. The direction of migration is shown by arrows. The mass markers are shown for the first 1D and second 2D dimension. L stands for lysate and pY-IP for phosphotyrosine immunoprecipitate.

Figure 7

WAVE1 and WAVE2 co-localize and dramatically change their distribution upon ephrinB1-Fc stimulation. Immunofluorescence analysis of the (−) cells (0 minutes) and cells (+) for 5, 10, 20 and 45 minutes with EphB1-Fc. The cells were analyzed for the presence of Actin (blue), WAVE1 (green) and WAVE2 (red) proteins. Scale bar, 20 μm.