Discovery of effectors for casein kinase signaling in the African trypanosome

Extensive divergence of protein sequences from those of model eukaryotes makes recognition of well-studied signaling pathways impossible in Trypanosoma brucei. Consequently, substrates and effectors of most protein kinases are unknown. We addressed these general problems by developing workflows to (i) identify effectors for protein kinases, (ii) discover new effectors and substrates of CK1.2 as proof of principle, and (iii) decode linear motifs in CK1.2 substrates. Casein kinase CK1.2 promotes cytokinesis and division of mitochondrial DNA (kinetoplast) (kDNA). Applying our principles to CK1.2 we identified four new substrates, BBP59, LRRP1, CAP64, and BBP268, that harbored two unique linear motifs, S-x(1,3)-T-x(1,4)-S-x(2)-A-x(2)-[AIV] and S-x(1,3)-T-x(1,4)-S. Ectopic expression of BBP59 inhibited cytokinesis and kinetoplast DNA division, like knockdown of CK1.2. Phosphorylation of BBP59 was blocked after knockdown of CK1.2 or treatment with SB-431542 an inhibitor of CK1.2 (IC₅₀ 49.2 nM). Highlighting phospho-regulation of its activities, Ser72 substitution with Ala72 or Asp72 in BBP59 caused preferential blockage of cytokinesis or kDNA division. The sequence-agnostic workflow used to identify effectors of CK1.2 can be deployed for other protein kinases, and to assist functional annotation of hypothetical proteins.