Clonado y caracterización de quinasas relacionadas con la CDC2 en el parásito Trypanosoma cruzi


Gómez, Eliana Beatriz
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tesis doctoral
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Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales
Biblioteca Digital (UBA-FCEN)
Two CDC2-related protein kinase genes (crk), tzcrk3 and tzcrk1, were cloned from the parasite Trypanosoma cruzi. Recombinant proteins were used to raise specific antisera. Antibodies anti-TzCRK1 recognized a protein of 33 kDa in the three forms of the parasite. This antiserum showed that TzCRK1 is localized in the cytosol, kinetoplast and nucleus, and is constitutiver expressed though the cell cycle. Purified anti-TzCRK1 IgGs, immunoprecipitated a kinase activity which phosphorylated histone H1 and Rb. The recombinant GST-TzCRK1 had high in vitro kinase activity in the absense of regulatory proteins. CDC2 and CDKZ specific inhibitors could not abolish the TzCRK1 kinase activity. Transfection of COS-7 cells with tzcrk1 demonstrated for the first time that a CRK protein can bind mammalian cyclins; TzCRK1 co-immunoprecipitated with cyclins E, D3 and A suggesting a role for this kinase in cell cycle control. The TzCRK3 antisera recognized a 40 kDa protein in membrane and nuclear fractions and a 54 kDa protein in the citosolic fraction. The p13SUC1 protein coprecipitated a kinase activity from the citosol of the epimatigote stage. Yeast complementation assays showed that these T. cruzi CRKs, could not rescue the yeast CDC28 function. The two hybrid system was used to identify and clone proteins that associate to TzCRK1. Three clones were identified, which code for proteins with identity to cyclins from the PREG and PHo80 family. In other organisms, these cyclin like proteins are involved in the control of phosphate metabolism. In S. cerevisiae, PH080 binds to the CDK protein PHO85, which controls the transcription of the acid phosphatase gene and participates in the regulation of the cell cycle. These results suggest that TzCRK1 could be involved in cell cycle progression and/or in other processes such as phosphate metabolism.
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