Cbk1 Exhibits Phosphorylation Site Preference for a Basic Motif with His at the −5 Position

<div><p>(A) Cbk1 kinase domain purified from E. coli was incubated with the indicated array of biotinylated peptide libraries in the presence of γ-<sup>32</sup>P-ATP, transferred to a strepavidin membrane, and kinase activity was analyzed by autoradiography. Numbers at the top of each column indicate positions relative to the phosphoacceptor S/T residue; rows contain specific amino acids in those positions. The optimal Cbk1 consensus sequence is HXRRX(S/T) as determined by quantification of the most highly phosphorylated peptide pools in the array.</p> <p>(B) Schematic diagram of Ace2 protein showing location of Cbk1 phosphorylation consensus sequences.</p> <p>(C) In vitro phosphorylation of N-terminally GST-tagged Ace2 fragments by immunoprecipitated Cbk1-HA. Combined Ala substitution of residues S113, S122, and S137 abolished <sup>32</sup>P labeling of the Ace2 fragment (lane 4). His at position 117 is required for phosphorylation of S122 (lane 5). Protein levels for GST-Ace2 and Cbk1-HA were confirmed to be similar for all reactions by immunoblotting against GST and HA. WT, wild-type.</p></div>