Label-free, quantitative analysis of PKA-dependent phosphoproteome changes in Trypanosoma brucei

Protein kinase A (PKA), the main effector of second messenger cAMP, is highly conserved among eukaryotes and a paradigm for the mechanisms of regulation of protein kinases by ligands. The unique PKA holoenzymes in the phylogenetically distant protozoan parasite Trypanosoma are unresponsive to cAMP in vitro and in vivo. By small molecule screening and optimization, we designed direct, membrane-permeable activators binding with a kD of 9 nM to the CNB pockets of the T. brucei regulatory PKA subunit. 7-Cyano-7-deazainosine has low toxicity and thus is a perfect tool to explore cAMP-independent PKA signaling in these important pathogens. This project describes PKA-induced phosphoproteome changes of the bloodstream stage of T. brucei.