The work was arranged in 3 work packages (WP)
WP1: Is YAK kinase translocated to the nucleus or within the cytoplasm upon reception of the SIF signal and is this mediated through interaction with 14-3-3 class proteins?
We had already created a YAK null mutant, this providing a suitable genetic background to introduce mutant YAK kinase forms to address location, interactions and function of the molecule. However, we found that the expression level of the kinase was very low, undetectable by western blot and immunofluorescence. This was also true when 'overexpression' was attempted, suggesting that the protein level of the kinase is tightly controlled. This prevented us from analysing the localization of the protein and selection of the protein to identify its interaction partners. Therefore, other strategies were used to analyse interactions and downstream target/substrates, for example through analysis of the YAK kinase null mutant for changes in their expressed proteins or levels of phosphorylation, this modification often controlling the activity of proteins (see WP3).
WP2: What is the kinase specificity of YAK kinase in vivo and in vitro?
To analyse the activity of YAK kinase, we first expressed the molecule in bacteria. However, activity assays with the recombinant protein were unsuccessful due to the conditions required for its solubilisation. We therefore used an insect cell expression system to produce the recombinant protein, either in its wild type form or where it had been mutated on different domains predicted to be important in its function. We evaluated several substrates of which the best was then used to determine the activity of the kinase mutated on key residues. The experiments revealed the essential role of residues in the catalytic domain of the protein as well as the importance of potential phosphorylation in the N-terminal domain, and the N-terminal domain itself. Also, one potential phosphorylation site in the N-terminal domain seemed to be implicated in the inhibition of kinase activity, whereas a trypanosome-specific insert present in the catalytic domain was essential for activity.
WP2 also investigated the role of the kinase, and predicted kinase activity in parasites. Due to the low expression of the protein, we overexpressed the kinase, and mutant forms of it, in trypanosomes and analysed parasite growth and their response to a chemical mimic of the SIF signal. This identified essential residues or domains for activity. In the absence of protein data, we quantitated the mRNA level of the kinase, this establishing that the YAK mRNA level is dependent on its predicted kinase activity, suggesting a feedback control loop.
In vivo we demonstrated that YAK kinase expression drives the differentiation from slender to stumpy forms of the parasites at a lower density than observed in control samples, confirming the participation of the kinase in the density-sensing developmental response.
WP3: What substrates are regulated by YAK kinase to generated developmental responses?
Initially, an evolutionary analysis was performed on the CMGC-class of protein kinases (which includes the YAK kinase) from different organisms, including trypanosomes. This revealed that the predicted YAK kinase belongs to a divergent branch of the DYRK sub-family. To investigate the potential (direct or indirect) interactants/substrates of the kinase, the complete profile of phosphorylated proteins of a YAK kinase null mutant was compared to wild type cells using mass spectrometry. This revealed a set of proteins that were reproducibly regulated (more or less abundant/phosphorylated) in the absence of the kinase. Molecules that are possible substrates of the kinase (i.e. less phosphorylated in the null mutant) are being interrogated for potential function in parasite differentiation.
In parallel, to better understand the QS pathway a new genetic screen was performed using a small compound identified in a drug screen, which drives stumpy formation. Currently, four candidates from the screen and their effects on resistance to the small compound in vitro and on stumpy differentiation in vivo are being tested. Our preliminary experiments have found that two of these genes act in stumpy differentiation and the relationship of these molecules to the operation of the YAK kinase is being explored.
