Fig. 5: Motif and upstream kinase analysis of regulated phosphosites identifies RXXS/T-directed AGC kinases and proline-directed CMGC kinases as mediators of EphA2 signaling.

a Enriched motifs were identified using the Motifs Analysis Tool from PhosphoSitePlus. 13-mer phosphosite-centered peptide sequences of regulated phosphosites were analyzed using the Motif-All algorithm (significance threshold of 1e⁻⁰⁵, support threshold of 0.05). A minimum motif occurrence threshold of five was manually applied to significantly overrepresented motifs. Motif logos were generated from the sequences of all 128 regulated phosphosites and from the phosphosites conforming with a particular enriched motif as indicated. Schematics showing proteins with an EphA2-regulated (RX)RXX[S/T]* AGC kinase motif (b) and [S/T]*P proline-directed CMGC kinase motif (c) organized by cellular functions. Phosphopeptide motifs are indicated by colored squares; regulation (up/down) is indicated by red and green arrows. d For the analysis of kinase-substrate relationship the PhosphoSitePlus, HRPD, Uniprot and NetworKIN databases were queried for known and predicted upstream kinases of regulated phosphorylation sites. Resulting kinase-substrate relationships are shown as black edges for known connections and as blue edges for connections predicted by the NetworKIN database. Kinases are organized according to kinase families. Only kinases with at least 3 substrate connections are shown. For the full network see Supplementary Fig. 5c. CMGC kinases are primarily proline-directed kinases. AGC kinases comprise kinases with a substrate specificity for RXX[S/T]* motifs. Phosphorylation sites with an RXX[S/T]* or an [S/T]*P motif are indicated by red and blue outlines around phosphosite nodes, respectively. Kinase node sizes reflect the number of regulated phosphosite connected to a kinase. mTOR, AKT, ERK, GSK-3β and RSK are the most likely candidate kinases for mediating signaling downstream of EphA2.