The protozoan parasite Trypanosoma brucei is the causative agent
of human African sleeping sickness and related animal diseases, and it has over
170 predicted protein kinases. Protein phosphorylation is a key regulatory
mechanism for cellular function that, thus far, has been studied in
T.brucei principally through putative kinase mRNA knockdown
and observation of the resulting phenotype. However, despite the relatively
large kinome of this organism and the demonstrated essentiality of several
T. brucei kinases, very few specific phosphorylation sites
have been determined in this organism. Using a gel-free, phosphopeptide
enrichment-based proteomics approach we performed the first large scale
phosphorylation site analyses for T.brucei. Serine, threonine,
and tyrosine phosphorylation sites were determined for a cytosolic protein
fraction of the bloodstream form of the parasite, resulting in the
identification of 491 phosphoproteins based on the identification of 852 unique
phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in
this study are predicted from their genome annotations to participate in a wide
variety of biological processes, including signal transduction, processing of
DNA and RNA, protein synthesis, and degradation and to a minor extent in
metabolic pathways. The analysis of phosphopeptides and phosphorylation sites
was facilitated by in-house developed software, and this automated approach was
validated by manual annotation of spectra of the kinase subset of proteins.
Analysis of the cytosolic bloodstream form T. brucei kinome
revealed the presence of 44 phosphorylated protein kinases in our data set that
could be classified into the major eukaryotic protein kinase groups by applying
a multilevel hidden Markov model library of the kinase catalytic domain.
Identification of the kinase phosphorylation sites showed conserved
phosphorylation sequence motifs in several kinase activation segments,
supporting the view that phosphorylation-based signaling is a general and
fundamental regulatory process that extends to this highly divergent lower
eukaryote.
