Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.

Phosphorylation sites A B

FGF4 phosphoproteomics analysis in mESCs.
A) Fgf4-/-mESCs were treated with 10 μM SU5402 for 1 h and stimulated with FGF4 for the indicated times. ppERK1/2, AKT pS473, GSK3B pS9 and tubulin levels were determined by immunoblotting. B) Venn diagrams showing the overlap between the phosphoproteomics data generated in this study and two previous large-scale phosphoproteomics datasets from FGF-stimulated ESCs. Source data are provided as a Source Data file.

EPHA2 is the major EPH receptor expressed in mESCs.
A) Average protein copy number per cell for EPH receptor family members in mESCs using quantitative whole-cell proteomics. Data are presented as mean ± SD (n=3) B) mRNA expression of EPH receptor family members in mESCs determined by qRT-PCR analysis. Data presented as technical replicates of a representative experiment. C) mRNA expression of Epha1 in Epha2+/+, Epha2-/-or Epha2-/-mESCs (clone C4) stably expressing EPHA2 was determined by qRT-PCR analysis. Data presented as technical replicates from a representative experiment. Source data are provided as a Source Data file. Epha2-/-mESCs EPHA2 supports mESC pluripotency and restricts commitment to differentiation.

EPHA2 inhibitory phosphorylation and transcriptional suppression during mESC differentiation.
A) Epha2-/-mESCs were transfected with either empty vector or increasing amounts of EPHA2 and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated and pTyr and EPHA2 levels determined by immunoblotting. B) Epha2-/-mESCs were transfected with either empty vector or indicated EPHA2 expression vectors and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated and pTyr and EPHA2 levels determined by immunoblotting. C) Epha2+/+ mESCs were differentiated as embryoid bodies (EBs) for 10 days in the presence of vehicle control, 1 μM PD0325901 or AZD4547, and levels of Fgf5 (top) and Brachyury (bottom) mRNA determined by qRT-PCR. Fold change in mRNA expression compared to control is presented as mean ± SD (n=4). statistical significance was determined using unpaired two-sided Student's t-test comparing each group to the control (Fgf5 **P = 0.019, ****P < 0.0001, Brachyury **P = 0.0027, *P = 0.0211) D) EPH receptor mRNA expression was determined by qRT-PCR in mESCs or EBs differentiated for 6 days. Fold change of expression between EBs and mESCs is presented from two independent experiments. E) Fgf4-/-mESCs were differentiated in the presence FGF4 for the indicated times, and EPHA2, OCT4, ERK1/2 and CDH1 levels determined by immunoblotting. F) 2i mESCs were differentiated in N2B27 medium for the indicated times and EPHA2, OCT4, and CDH1 levels were determined by immunoblotting. G) 2i mESCs were treated with 2 μM of the indicated inhibitors and differentiated in N2B27 medium for 3 days. EPHA2, NANOG, ERK1/2 and tubulin levels were determined by immunoblotting. Source data are provided as a Source Data file.

EPH receptor expression during a time-course of early mouse embryonic development
Data corresponding to EPH receptor mRNA expression was extracted from Table S1      Epha2 exon 3

Generation of CRISPR/Cas9
Epha2-/-cell lines A) Epha2-/-mESCs were generated using CRISPR/Cas9 D10A and the indicated sense and antisense gRNA sequences as described in experimental procedures. Genomic and predicted protein sequences of 3 independent Epha2-/-clones were determined. B) Schematic of the predicted Epha2 gene and protein in Epha2-/-clones. C) Immunoblot analysis of EPHA2 expression using N-terminal and C-terminal specific antibodies in control wild-type mESCs and prospective Epha2-/-mESC clones. mESC lines that were selected for further analysis and experiments are indicated.    Figure 9: Full-size blot scans

Phosphoproteomic profiling
Cell culture and proteomic sample preparation: Fgf4-/-mESCs were cultured in LIF/FBS then starved  with a 1% FDR using Percolator.
The phosphoRS 6 node was used to localize phosphorylation sites with a probability at least 90% to be considered as confident. Both unique and razor peptides were used for quantitation.
Reporter ion abundances were corrected for isotopic impurities based on the manufacturer's data sheets. Signal-to-noise (S/N) values were used to represent the reporter ion abundance with a coisolation threshold of 50% and an average reporter S/N threshold of 10 and above required for quantitation from each MS2 spectra to be used. The S/N value of each reporter ion from each PSM were used to represent the abundance of the localised phosphorylation sites. The precursor spectra with higher than 25% co-isolation were checked manually. The median was used for the normalisation and quantification calculated as average of three biological replicates, with standard deviation of three biological replicates lower than 25% used for further analyses.