Fig. 7. Multiple kinases phosphorylate the five serine/threonine residues in the EphA2 kinase-SAM linker.

a Screen of 298 kinases using in vitro kinase reactions with [γ-³³P] ATP and a peptide substrate containing S892, T898, S899, S901 and phosphorylated S897 identifies many kinases that phosphorylate residues other than S897. The 44 kinases (15% of those screened) mediating the highest ³³P incorporation are shown in red. b The 24 kinases mediating the highest ³³P incorporation (>50,000 CPM) are shown in red and other less active family members that were also tested are shown in gray. Kinase families are ordered based on the most active family member. c CK1 family members preferentially phosphorylate the peptide with phosphorylated S897 compared to two peptides with Ala replacing S897 and differing in the last residue (averaged together). d Comparison of two peptides containing S892 and differing in the last residue (averaged together) with a peptide in which S892 is replaced by Ala shows that many of the kinases identified in the screen mainly phosphorylate S892. Kinase families are ordered as in b. e Comparison of the phosphorylation of the two indicated peptides identifies kinases that can phosphorylate T898 and/or S899 in the presence or absence of prior S892, S897 and S901 phosphorylation. f ³³P incorporation into peptides in which only S897 can be phosphorylated identifies kinase families that can phosphorylate this residue. Higher ³³P incorporation into the peptide also containing the other four serine/threonine residues and residual ³³P incorporation into the peptide with already phosphorylated S897 suggest that some of the kinases can also phosphorylate other residues. Individual data points in c and d (for the measurements with two peptides differing only in the last residue) and in e (for duplicate measurements with CK2 kinases, PLK3, MAP3K9 and CAMK2B) are shown as gray dots. CPM, counts per minute measuring incorporated ³³P; RLU, relative light units. Graphs were generated using Prism software (GraphPad). Source data for the in vitro kinase reactions are provided in Supplementary Table 3.