IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
New insight into wee1 protein kinase homologue from Trypanosome brucei
Autor/es:
BOYNAK, N; ROJAS, F; D'ALESSIO, C; RODRIGUEZ, V; GHIRINGHELLI, D; TÉLLEZ-IÑÓN, MT
Lugar:
Ascochinga, Córdoba
Reunión:
Congreso; XXIV Reunión Científica Anual de la Sociedad Argentina de Protozoología; 2010
Institución organizadora:
Sociedad Argentina de Protozoología
Resumen:
The cell cycle of eukaryotic cells is coordinated by cyclin-dependent protein kinases (CDKs) that are activated by association with specific cyclin proteins. Activation of the complex cyclin B- cdc2 kinase is a pivotal step in mitotic initiation. Phosphorylation of cdc2 in the Tyr15 inhibitory site by the Wee1 kinase suppresses the cdc2 activity during interphase. In several organisms, homologues of Wee have been identified. The trypanosomatids are classified as some of the most divergent eukaryotes, and a large gene family of cdc2-related kinases (CRKs) and cyclins has been identified in these organisms. However, despite the high amino acidic conservation in the key regulatory positions compared with the mammalian CDKs, the regulation of CRK activity and the mechanisms that underlie the control of the cell cycle and the differentiation processes are still under investigation in these parasites. Searching the proteins that could control the G2/M stage in the Trypanosoma genome database (GeneDB), we have recently identified a wee1 orthologue in the T. brucei genome (TbWee). The essential role of TbWee for cell cycle progression was shown by RNAi technique. To evaluate if TbWee gene is able to rescue the Schizosaccharomyces pombe mutant, S. pombe wee1-50 strain was transformed with either the pREP3x vector alone, pREP3x-S. pombe wee1 or the pREP3x–TbWee. Cells transformed with pREP3x grew normally whereas overexpression of TbWee caused the cells to increase in size indicating partial rescue of the mutant. To further characterize TbWee, we purified the recombinant protein TbWee1-His obtained from Sf9 insect cells and used in kinase assays. Phosphorylation of histone H1 and poly glu- tyr as substrates, were not detected. Autophosphorylation of TbWee on tyrosine residues was shown in Western blot. Using a modified in-gel kinase assay we observed that Tbwee has enzymatic activity. Hence TbWee exhibits functional properties that are characteristic of wee kinases. Reverse genetics is being carried out to identify the function of this kinase in the cell cycle of these eukaryotic cells.