RESUMO
Trypanosoma cruzi ribosomes from epimastigote forms were purified as determined by electron microscopy and isoelectrofocusing was used to analyse this purified ribosome fraction. Silver stained gels revealed that acidic proteins are present in at least 10 different isoforms, in accord with previous cloning studies. To detect phosphorylation, in vitro phosphorylation assays using the recombinant protein TcP2beta-mbp were carried out. The results showed that T. cruzi cytosolic fraction possesses protein kinase activity able to phosphorylate the recombinant protein. Purified ribosomes contain protein kinases that could also phosphorylate the recombinant protein TcP2beta-mbp. Labelling parasites with [(32)Pi] in a phosphate free medium demonstrated that ribosome proteins, recognised with a specific mouse antiserum against recombinant TcP2beta proteins, are phosphorylated in vivo. All these results suggest that in vivo phosphorylation of ribosome TcP2beta proteins are mediated by protein kinase(s) not yet identified.
Assuntos
Proteínas de Protozoários , Proteínas Ribossômicas/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Microscopia Eletrônica , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/química , Ribossomos/química , Ribossomos/metabolismoRESUMO
Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.
Assuntos
Ciclinas/isolamento & purificação , Proteínas Quinases/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2 , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/isolamento & purificação , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Citoplasma/enzimologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Histonas/metabolismo , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Cinetina , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Piperidinas/farmacologia , Testes de Precipitina , Proteínas Quinases/isolamento & purificação , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Purinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Alinhamento de Sequência , Trypanosoma cruzi/metabolismoRESUMO
Two cdc2-related protein kinases (crk), tzcrk3 and tzcrk1, from the protozoan parasite Trypanosoma cruzi were cloned. tzcrk3 encodes a 35 kDa protein sharing 51.5% amino acid identity with human cdc2 and 82% identity with Trypanosoma brucei CRK3. tzcrk1 encodes a 33 kDa protein sharing 52.7% identity with human cdc2 and a high degree of identity (> 78%) with T. brucei CRK1, Leishmania mexicana CRK1 and Trypanosoma congolense CRK1. A recombinant TzCRK1 protein was able to phosphorylate histone HI and retinoblastoma protein. Western blotting using a polyclonal antibody raised against the recombinant TzCRK1 protein showed that the kinase is present in all life cycle stages of the parasite. A PSTAIRE antiserum detected proteins of 32, 33 and 35 kDa, with differential expression in the life cycle of the parasite. 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. These results indicate that T. cruzi might have cyclin homologues that control the activity of the CRK proteins and that a complex mechanism would exist in order to regulate the kinases involved in the cell cycle and the differentiation processes of the parasite.