Characterization of the Trypanosoma cruzi Cdc2p-related protein kinase 1 and identification of three novel associating cyclins

Several Cdc2p-related protein kinases (CRKs) have been described in trypano somatids but their role in the control of the cell cycle nor their biologic al functions have been addressed. In Trypanosoma cruzi two CRKs have been i dentified, 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 c ycle, and show that it is localized in the cytoplasm, discrete regions of t he nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplas t), suggesting a putative control function in this organelle. Using purifie d anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cru zi epimastigote forms a protein kinase activity which is not inhibited by C DK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomouci ne. Lastly, using the yeast two-hybrid system we identified three novel cyc lin-like proteins able to associate with TzCRK1. and demonstrate that two o f these cyclins also bind the T. cruzi CRK1 protein, indicating that these two CRKs are cyclin-dependent kinases. (C) 2001 Elsevier Science B.V. All r ights reserved.