Protein kinase B (PKB) is an enzyme that assists cells to proliferate under stressful conditions. In human cells, aberrant PKB activity is implicated in many cancers, making PKB an important target for further research. We are studying PKB in Dictyostelium, a social amoeba that has highly conserved protein kinase B signaling pathways similar to those in human cells. In Dictyostelium, PKB is activated when cells are under nutrient-poor conditions. To activate PKB in the laboratory, the cells are starved to initiate the development cycle and pulsed with a signaling compound, cAMP, as the first messenger. A fluorescently labelled peptide reporter, VI-B, is a peptidase-resistant synthetic substrate of PKB that can be utilized to measure the activation of PKB by capillary electrophoresis with laser-induced fluorescence (CE-LIF). Although VI-B was developed in human cell lines, we are validating it to use in Dictyostelium. Though background phosphorylation occurs in vegetative control cells, the data suggest that PKB is activated by cAMP pulsing and the phosphorylation by PKB is specific to development. Physiological, genetic and pharmaceutical control experiments are in progress to confirm the specificity of the peptide reporter for PKB.

Tracking PKB activity during Dictyostelium development using a peptide reporter

252nd National Meeting (2016)

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