Biological and biochemical characterization of the extracellular signal-regulated kinase 8  homolog (TbERK8) in Trypanosoma brucei

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Virginia Tech


Trypanosoma brucei species are vector-borne protozoan parasites that cause Human African typanosomiasis (HAT) and nagana in cattle. In humans, the diseases caused by these parasites are fatal if left untreated. Treatments for these diseases are complicated because the approved drugs for treatment are ineffective against the parasites and have many toxic side effects associated with their use. There is a clear need to identify new therapeutics that are less toxic and more effective against T. brucei. Our approach for identifying new therapies is to identify novel targets in the parasite that can be modulated by small molecules. The mitogen-activated protein kinases (MAPK) pathway is a three-tiered signaling cascade that regulates cell responses to stimuli and are involved in essential processes. MAPKs can regulate differentiation, virulence, apoptosis, cell cycle and gene expression, which makes MAPKs interesting drug targets in T. brucei. The extracellular-signal regulated kinase 8 homolog in T. brucei (TbERK8) is essential for survival in bloodstream form T. brucei. The work in this dissertation involves characterizing this T. brucei MAPK to better understand its biological function and identify small molecules that can inhibit its activity to kill the parasite. Here, we report that TbERK8 is an atypical MAPK kinase that is able to autophosphorylate and no upstream kinases that activate TbERK8 have been identified. We have demonstrated that TbERK8 is able to phosphorylate the proliferating cell nuclear antigen homolog in T. brucei (TbPCNA). This is in contrast to the reported function the human ERK8 and PCNA homologs that form a stable complex in normal breast cells which does not result in PCNA phosphorylation. We also report here that TbPCNA is phosphorylated on three residues localized to a unique insertion loop by TbERK8. TbPCNA is tightly regulated in the parasites such that either upregulating or downregulating its expression arrests T. brucei proliferation. Although, this mechanism of phosphorylation is unique to TbPCNA, the role that such phosphorylation has in regulating TbPCNA is not known. Finally, we have identified small molecules that can selectively inhibit either TbERK8 or HsERK8, demonstrating that TbERK8 can be selectively inhibited to kill the parasite. The unique properties of TbERK8 can be exploited by small molecules that can be developed into new parasite-specific therapies that kill T. brucei with fewer side effects to the patients.



Trypanosoma brucei, Extra-cellular signal regulated kinase 8 (ERK8), Proliferating cell nuclear antigen (PCNA), PIP-box, phosphorylation