Cbk1 Exhibits Phosphorylation Site Preference for a Basic Motif with His at the −5 Position
(A) Cbk1 kinase domain purified from E. coli was incubated with the indicated array of biotinylated peptide libraries in the presence of γ-32P-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.
(B) Schematic diagram of Ace2 protein showing location of Cbk1 phosphorylation consensus sequences.
(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 32P 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.
