Fig. 7: RON4 is a direct substrate of RON13.

a Partial view of the RON13 structure highlighting the autophosphorylation sites (arrows, red). Kinase domain (orange tones). NLI (N-lobe insertion). b Schematic representation of RON13 structure organization highlighting the autophosphorylated residues (red) mutated in this study. The kinase domain and the C-terminal extension (CTE) are colored in orange and gray tones. c Radioactive kinase activity assays comparing RON4 phosphorylation and RON13 autophosphorylation for different rRON13 phospho-mutants; phosphonull (RON13pn) and phosphomimetic (RON13pm). Autoradiograph of a representative radioactive kinase assay is shown below the graph. Data shown as mean ± SD normalized to wt (n = 3 biologically independent experiments). P values were determined using a two-tailed paired t test. d Radioactive kinase activity assays characterizing the influence of ions on rRON4 phosphorylation by RON13. Densitometry data (mean ± SD; n = 3 biologically independent experiments) were normalized to RON4 phosphorylation in presence of Mn²⁺. Statistical significance was determined using a two-tailed paired t test. e Autoradiograph from a radioactive kinase activity assays in presence of MBP (left) or RON4 (right) as a substrate. f Schematic representation of the two states analyzed by HDX-MS. The kinase domain and the C-terminal extension (CTE) are colored in orange and gray tones. NLI (red). RON4 (violet). g Differences in HDX rates between rRON13dk alone and in the presence of RON4. A single region encompassing amino acids 377–390 is protected by RON4, indicating the contact site between the two proteins. Data shown as mean ± SD; n = 3 biologically independent experiments. h RON13 kinase domain highlighting the RON4-binding site (residues 377–390; blue) identified by HDX-MS. This region corresponds to the NLI and encompasses the two phosphosites Thr379 and Ser381. Source data are provided as a Source data file.