Systems-level analysis of RDK1 reveals compartment-specific kinase activity and a function in the maintenance of the mitochondrial proteome in Trypanosoma brucei
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Repressor-of-differentiation kinase 1 (RDK1) is one of two kinases expressed in bloodstream form Trypanosoma brucei parasites that were found to repress premature and spontaneous differentiation into the insect procyclic form. However, the effect of RDK1 RNAi was previously limited to the expression of a single surface coat protein, EP1 procyclin. Thus, there remains a significant gap in knowledge on the impact of RDK1 expression in bloodstream form T. brucei parasites. Here, we employ a systems biology approach and performed several proteomics analyses to identify RDK1 protein interactions and to determine the impact of loss of RDK1 expression on the bloodstream form proteome and phosphoproteome to uncover clues about potential mechanisms for RDK1 function. We found that RDK1 is dual localized to the cell membrane and the mitochondrial inner membrane with the kinase domain oriented towards the cytoplasm and mitochondrial inner membrane. Unexpectedly, the most enriched RDK1-proximal proteins were mitochondrial proteins. Furthermore, RDK1 depletion causes bloodstream form parasites to significantly upregulate many mitochondrial proteins and glycosomal proteins, several of which are upregulated in procyclic form parasites. Surprisingly, the mitochondrial phosphoproteome is largely unaffected by RDK1 depletion, while RDK1-dependent phosphoregulation is restricted to the cell membrane localization of RDK1. Lastly, we determined that RDK1 does not possess adenyl cyclase activity or alter intracellular cAMP levels; however, the dysregulated phosphoproteins correlate with functions in cyclic nucleotide signaling. In conclusion, RDK1 exhibits localization-specific kinase activity to regulate cyclic nucleotide signaling and mitochondrial proteomic maintenance in bloodstream form parasites.
IMPORTANCE
Trypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock across 36 sub-Saharan African countries. The parasite encounters different environmental niches as it is transmitted from an infected human to the tsetse fly vector as the fly takes a blood meal. T. brucei must sense environmental cues to initiate intracellular signaling pathways to promote effective differentiation and cellular remodeling from the mammalian bloodstream forms to the insect procyclic form. RDK1 is one of two kinases shown to repress premature differentiation to procyclic form, which would be detrimental for parasite survival in the human host. Therefore, it is essential to uncover mechanisms of RDK1 function to better understand how T. brucei maintains homeostasis in the human host and signals for effective cellular remodeling during parasite transmission.
