The MAP kinase MAPKLK1 is essential to Trypanosoma brucei proliferation and regulates proteins involved in mRNA metabolism.

Protein phosphorylation and dephosphorylation events regulate many cellular processes. The identification of all phosphorylation sites and their association to a respective protein kinase or phosphatase is a challenging and crucial step to have a deeper understanding of the effects of signaling networks on cells. Pathogenic trypanosomatids have a large number of protein kinases and phosphatases in comparison to other organisms, which reinforces the relevance of the phosphorylation process in these early eukaryotes, nevertheless little is known about protein phosphorylation in these protozoa. In this context, the role of a MAP kinase-like kinase (MAPKLK1), observed to be essential to proliferation of procyclic Trypanosoma brucei, was studied. After silencing MAPKLK1 expression by RNAi, the cells were evaluated by SILAC MS-based proteomics and RNA-Seq. We identified 1756 phosphorylation sites of which 384 were not previously described in T. brucei. Despite being essential, few modulations were observed at the phosphorylation patterns and gene expression levels of MAPKLK1 knockdown. These indirect targets and potential substrates of MAPKLK1 are related to key cellular processes enriched to mRNA processing and stability control.

SIGNIFICANCE: The field of cell signaling is a promising topic of study for trypanosomatids, since little is known about this topic and the gene expression regulation occurs at post-transcriptional level. In this sense, the present work increases the knowledge on protein phosphorylation process in Trypanosoma brucei. We depleted one MAP kinase (MAPKLK1) of T. brucei and evaluated the effects on the cell. We showed that MAPKLK1 is essential to the cell, while few modulations on phosphoproteome, proteome and transcriptome are observed with its depletion. Although in low number, the changes in phosphoproteome were significant, presenting possible substrate candidates of MAPKLK1 and indirect targets related to mRNA processing and stability control, metabolic pathways, among others. This result provides insights in the phosphorylation network of T. brucei, a model organism that impacts human and animal health.