EGFR Phosphorylates and Associates with EFNB1 to Regulate Cell Adhesion to Fibronectin

Abstract

Ephrin-Bs (EFNB1-3) are ligands for members of the largest subfamily of receptor tyrosine kinases (RTKs) in humans, the EPH receptors. Interestingly, ephrin-Bs are transmembrane proteins that may also act as receptors themselves upon EPH binding, activating so-called reverse signaling pathways that are critical for multiple cellular processes. Although a number of ephrin-B signaling effectors have been identified, the molecular mechanisms underlying ephrin-B-driven cellular processes remain unresolved, suggesting that multiple signaling effectors are yet to be discovered. Here, we employed proximity labeling proteomics to delineate the proximity network of EFNB1 in steady state and under active reverse signaling conditions. This allowed us to identify 90 uncharacterized EFNB1 proximity partners, from which we could distinguish three main groups: EPH receptor stimulation-dependent, stimulation-independent, and negatively modulated by EPH receptor stimulation. We further investigated the functional relationship between EFNB1 and one of the candidates identified, the epidermal growth factor receptor (EGFR). We found that EFNB1 and EGFR associate in cells and showed that the formation of this complex relies on EFNB1's PDZ-binding motif (PBM). Strikingly, we demonstrate that EGFR directly phosphorylates tyrosine residues within EFNB1's PBM, which results in the disruption of the EFNB1-EGFR complex. Furthermore, we show that the EFNB1-EGFR association is required for EFNB1-dependent cell adhesion to fibronectin. Taken together, our results shed light on a functional relationship between EFNB1 and EGFR.

Graphical abstract

Fig. 1

Proximity labeling proteomics reveals EFNB1’s steady state and EPHB3 stimulation-dependent networks.A, schematic representation of miniT-Flag fusions. The promiscuous biotin ligase miniT and a Flag tag were fused at the C-terminus of EFNB1 or YFP (control). B, expression of miniT-Flag constructs in HEK293 T-REx stable cell lines. Tetracycline (Tet) treatment induces expression of EFNB1-miniT-Flag or YFP-miniT-Flag fusions in HEK293 T-REx stable cell lines. C, biotinylation of proximal proteins by miniT-Flag fusions was assessed by western blotting using streptavidin-HRP. Addition of biotin to Tet-treated EFNB1-miniT-Flag or YFP-miniT-Flag stable cells lines leads to biotinylation of endogenous proteins. D, EFNB1-miniT-Flag expressing cells (HEK-EFNB1-miniT-Flag) were stimulated with EPHB3 expressing cells (HEK-EPHB3) for different times. EFNB1-miniT-Flag Tyr phosphorylation was assessed with an anti-phospho-EFNB antibody (pEFNB). Y/F mutants of EFNB1 (EFNB1-5F-miniT-Flag and EFNB1-6F-miniT-Flag) were used as negative controls. EFNB1-5F-miniT-Flag carries Y/F mutations at positions 317, 324, 329, 343 and 344. EFNB1-6F-miniT-Flag carries Y/F mutations at positions 313, 317, 324, 329, 343 and 344. E, subcellular localization of miniT-Flag fusions was analyzed by immunofluorescence using anti-Flag antibodies. EFNB1-miniT is enriched at the plasma membrane (white arrows) while YFP-miniT is distributed throughout the cells. Scale bar represents 20 μm. F, proximity network for EFNB1 in steady state and under EPHB3-stimulation (EFNB1+EPHB3). The network is divided into three main sections: (left) proximity partners that are negatively regulated by EPHB3-stimulation; (center) EPHB3-stimulation independent proximity partners and (right) EPHB3-stimulation dependent proximity partners. Only proteins identified with high confidence (Bayesian False Discovery Rate BFDR ≤1%) are displayed.

Fig. 2

EGFR associates with EFNB1 and is required for EFNB1-dependent cell adhesion to fibronectin.A, HeLa cells overexpressing EFNB1 (HeLa-EFNB1) were transfected independently with two siRNAs to evaluate the effect of targets’ depletion on EFNB1-dependent cell adhesion to fibronectin. Values were normalized relative to HeLa-EFNB1 cells treated with siCtrl (dashed red line). Error bars indicate SD (∗p < 0.05, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001; one-way ANOVA and Dunnett’s post-hoc test). B, Western blot analysis of EGFR and EFNB1 levels from HeLa-Ctrl or HeLa-EFNB1 cells treated with siCtrl or siEGFR (from A). Numbers (in %) represent a fraction of loading. C, schematic representation of GFP-tagged EFNB1 fusions; mutants lacking the PBM (aa343–346) (B1-dPBM-GFP) or deletion mutant lacking most of the intracellular domain (aa 269–346) (B1-dICD-GFP). D, Western blot analysis of endogenous EGFR following GFP-immunoprecipitation from HEK293T cells overexpressing B1-GFP or GFP alone. E and F, Western blot analysis of EGFR following GFP-immunoprecipitation using cell lysates of HEK293T cells overexpressing EGFR and EFNB1 GFP-tagged constructs. G, cell adhesion to fibronectin was evaluated for HeLa T-REx cell lines stably overexpressing untagged WT EFNB1 or deletion mutants lacking the PBM (EFNB1-dPBM). Values were normalized relative to HeLa-EFNB1 cells. Error bars indicate SD (∗∗∗p < 0.001; one-way ANOVA and Dunnett’s post-hoc test). H, EFNB1 Western blot from HeLa T-REx lysates used in panel G.

Fig. 3

EGFR phosphorylates tyrosine residues within EFNB1 PDZ-binding motif.A, in vitro radioactive kinase assay using recombinant intracellular domain (ICD) of EFNB1 (aa259–346) or EFNB1-6F, and recombinant kinase domain of EGFR. Incorporation of radioactive ³²P was assessed by autoradiography. B, schematic representation of the last 34 aa of EFNB1 C-terminus. Position of the six Tyr residues is indicated on the top. C, in vitro kinase assay on peptide arrays displaying all Tyr residues in the intracellular domain of EFNB1. Positions of the Tyr residues tested in each peptide are indicated on the top. The row on the top displays negative control peptides, where all Tyr resides were replaced by Phe (FF). Middle row (YF) displays peptides with a single Tyr residue available (marked in bold on top). Bottom row contains peptides with both Tyr residues available (YY). D, Full-length EFNB1 fused to GFP (B1-GFP) or Y/F mutants were co-overexpressed with EGFR. Activation of EGFR was evaluated with a phospho-EGFR antibody (pEGFR) and EFNB1 Tyr phosphorylation was assessed using the pTyr antibody 27B10. B1-6F-GFP contains Phe replacing all six Tyr of EFNB1; B1-2F-GFP refers to Y343, 344F.

Fig. 4

EFNB1 PBM Tyr phosphorylation disrupts its association with EGFR and blocks EFNB1-dependent cell adhesion to fibronectin.A and B, Western blotting of EGFR following GFP-immunoprecipitation from cell lysates of HEK293T cells overexpressing EFNB1 fused to GFP (B1-GFP) or EFNB1 phosphomimetic (Y/E (Y343, 344E); panel A) or non-phosphorylable (Y/F (Y343, 344F); panel B) mutants, and EGFR. C, cell adhesion to fibronectin was assessed for HeLa T-REx cell lines that stably overexpress WT-EFNB1 or phosphomimetic mutant EFNB1-2E. Values were normalized relative to HeLa-EFNB1. Error bars indicate SD (∗∗∗p = 0.0001, ∗∗∗∗p < 0.0001; one-way ANOVA and Dunnett’s post-hoc test), n = 3. D, EFNB1 Western blot from HeLa T-REx lysates used in panel C.