title: Extracellular-signal regulated kinase 8 of Trypanosoma brucei uniquely phosphorylates its proliferating cell nuclear antigen homolog and reveals exploitable properties.
abstract: The Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are vector-borne pathogens that cause sleeping sickness also known as Human African Trypanosomiasis (HAT), which is fatal if left untreated. The drugs that treat HAT are ineffective and cause toxic side effects. One strategy for identifying safer and more effective HAT drugs is to therapeutically exploit essential gene targets in T. brucei. Genes that make up a basic mitogen-activated protein kinase (MAPK) network are present in T. brucei. Tb927.10.5140 encodes an essential MAPK that is homologous to the human extracellular-signal regulated kinase 8 (HsERK8) which forms a tight complex with the replication factor proliferating cell nuclear antigen (PCNA) to stabilize intracellular PCNA levels. Here we demonstrate that (TbPCNA) is uniquely phos-phorylated on serine (S) and threonine (T) residues in T. brucei and that TbERK8 phosphorylates TbPCNA at each of these residues. The ability of an ERK8 homolog to phosphorylate a PCNA homolog is a novel biochemical property that is first demonstrated here in T. brucei and may be unique to this pathogen. We demonstrate that the potent HsERK8 inhibitor Ro318220, has an IC50 for TbERK8 that is several hundred times higher than its reported IC50 for HsERK8. This indicated that the active sites of TbERK8 and HsERK8 can be selectively inhibited, which provides a rational basis for discovering inhibitors that specifically target this essential parasite MAPK to kill the parasite.
authors: a Department of Biochemistry and Fralin Life Science Institute , Vector-Borne Disease Division, Virginia Polytechnic Institute and State University , Blacksburg , VA , USA.
authors: Ana L
authors: AL
authors: Valenciano
authors: b Department of Pharmaceutical Chemistry , University of California San Francisco , San Francisco , CA , USA.
authors: Giselle M
authors: GM
authors: Knudsen
authors: a Department of Biochemistry and Fralin Life Science Institute , Vector-Borne Disease Division, Virginia Polytechnic Institute and State University , Blacksburg , VA , USA.
authors: Zachary B
authors: ZB
authors: Mackey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010802:Phylogeny; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D061987:Sf9 Cells; D054852:Small Molecule Libraries / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology*
chemical_list: D007211:Indoles; D018809:Proliferating Cell Nuclear Antigen; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D048049:Extracellular Signal-Regulated MAP Kinases; C064758:Ro 31-8220
keywords: Trypanosoma brucei; evolution; extracellular-signal regulated kinase (ERK); homology modeling; inhibitors; mitogen-activated protein kinase (MAPK); proliferating cell nuclear antigen (PCNA); western blot