Evidence for CRK3 participation in the cell division cycle of Trypanosoma cruzi
Introduction
The parasitic protozoan Trypanosoma cruzi belongs to the Trypanosomatidae family and is responsible for the clinically important Chagas’ disease. Trypanosomes have a complex life cycle that alternates between insect and mammalian hosts with different developmental stages. The rapidly dividing forms such as amastigotes, in the vertebrate host cells, and epimastigotes, in the vector gut, maintain the infection. The non-dividing trypomastigote form, which is probably arrested in G1/G0 phase of the cell cycle, can be found in the vertebrate bloodstream or in the vector hindgut and is preadapted for survival when the parasite passes from one organism to the other [1], [2]. The coordination between replication and segregation of the nucleus, kinetoplast and flagellum is another important aspect of T. cruzi and other kinetoplastids cell cycle [3]. This parasite undergoes a complex series of morphogenetic changes suggesting that there is an integral link between control of the cell cycle and cell differentiation. Trypanosomes have gained complexity in cell cycle control and it is likely that special molecular mechanisms have evolved to meet special requirements of the parasite.
Cyclin dependent kinases (CDKs), such as Schizosaccharomyces pombe Cdc2p and Saccharomyces cerevisiae CDC28 are essential regulators of the cell cycle and are highly conserved, ubiquitous proteins throughout eukaryotes [4]. Control of cell cycle progression in both budding and fission yeast, is associated with the activation of a single CDK, CDC28 and Cdc2p, respectively. In higher eukaryotes, the regulation of the cell cycle is under the control of many CDKs. CDK levels tend to remain constant during the cell cycle, and their activities are controlled mainly post-translationally by different mechanisms: association of the kinase subunit with the positive regulatory cyclin partner, phosphorylation of conserved residues and binding of CDK inhibitor peptides [5], [6]. Over the last years considerable effort has been made to identify compounds that specifically inhibit CDKs (CKIs) to be used as anti-cancer agents. A number of chemical compounds have been identified and many of them appear to inhibit kinase activity by competitive binding to the ATP binding pocket. Some CKIs have remarkable selectivity and can differentiate the human CDK family into two subfamilies: (1) CDK1, CDK2 and CDK5, and (2) CDK4 and CDK6. Three chemical inhibitors, Olomoucine, Roscovitine and Flavopiridol have shown selectivity for CDK1 and CDK2 proteins [7], [8]. Flavopiridol (FP), a semi-synthetic flavone, is a potent growth inhibitor of a number of tumor cell lines [9], [10]; it blocks mammalian cells in either G1 or G2 [11] and it can also affect S-phase progression in Plasmodium falciparum [12]. In addition, it was found that Flavopiridol significantly inhibited glycogen phosphorylase a and b from rabbit skeletal muscle [13].
Investigations into the molecules that regulate the cell cycle of trypanosomatids have led to the identification of many proteins belonging to the Cdc2p related kinase (CRK) family. In T. cruzi we have isolated two CRK genes: TzCRK1 and TzCRK3 [14], [15]. CRK1 homologues have been found in the trypanosomatids Leishmania mexicana [16], Trypanosoma brucei [17] and Trypanosoma congolense (acc.# Z30312). Gene disruption experiments in L. mexicana indicated that CRK1 was essential to the promastigote form [18]. CRK1 proteins have been tested for their ability to complement Cdc2p/CDC28 mutations in yeast, but under the assayed conditions none of them could rescue the deficient phenotype [14], [16]. T. cruzi CRK3 predicted aminoacid sequence [14] has 82, 78 and 77% identity with T. brucei [17], L. mexicana [19] and L. major [20] CRK3, respectively. Genetic manipulation showed that CRK3 is essential to L. mexicana promastigotes [21]. There have been reports indicating that CRK3 could be the CDK1 functional homologue in Leishmania [19], [20], [21], [22].
In this study we report the characterization of T. cruzi CRK3 and show evidence indicating that this enzyme could be the cdk1 homologue in T. cruzi.
Section snippets
Cellular cultures and protein preparations
T. cruzi epimastigotes from Tul 2 strain were cultured as described [23]. Metacyclic trypomatigotes were obtained by axenic culture under differentiating conditions. Amastigotes were obtained from Vero cell cultures as described in [24].
Parasite protein extracts were prepared by resuspending the parasite pellets in SK buffer with proteinase inhibitors (0.25 M sucrose, 5 mM KCl, 0.5 mM N-Tosyl-l-lysine chloromethyl ketone, 1 mM benzamidine, 1 mM phenylmethyl-sulphonyl fluoride, 25 U ml−1
Stage specific Northern blot analysis of the CRK3
Total RNA isolated from three life cycle stages of T. cruzi was Northern blotted using full length CRK3 as hybridization probe (Fig. 1). As previously reported for the epimastigote form [14], CRK3 mRNA was detected as a band ranging between 1.3 and 1.4 kb. An approximate 2-fold increase in CRK3 mRNA levels was detected in amastigotes in three independent experiments (Fig. 1). The same Northern blot was re-hybridized with a ribosomal probe and the loaded RNA was normalized. There are at least
Discussion
In this work we report the characterization of Trypanosoma cruzi CRK3 and show evidence indicating that this protein kinase could be the CDK1 homologue in this parasite.
Western blot analysis using CRK3 antiserum revealed in epimastigote extracts a band of predicted molecular weight. Since the depleted CRK3 antiserum did not detect any signal we can confirm the specificity of the antibodies. Amastigote and trypomastigote stages showed bands of higher molecular weight (ca. 46 kDa). These bands
Acknowledgments
We are indebted to Dr. Berta Franke de Cazzullo from the National University of General San Martı́n, for providing T. cruzi amastigotes and trypomastigotes cultures. This work was supported by grants from the National Research Council (CONICET), the World Health Organization Special Program for Research and Training in Tropical Diseases, Ministerio de Salud and the University of Buenos Aires (UBA). M.I. Santori is a Doctoral Fellow of UBA; S. Ları́a is a Doctoral Fellow of Ministerio de Salud;
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Trimethylation of histone H3K76 by Dot1B enhances cell cycle progression after mitosis in Trypanosoma cruzi
2020, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :Recently, Hayashi and Akiyoshi [65] showed that introducing a proteasome-resistant version of CYC6 into T. brucei that interacts with cyclin-related kinase 3 (CRK3) and corresponds to mitotic cyclin B2 in other organisms arrests the cell cycle in late mitosis. In T. cruzi, CRK3 is also expressed when the parasite enters mitosis [66], and prevention of CYC6 degradation by proteasome inhibitors and overexpression of CYC6 arrest parasite division at the G2/M boundary [67]. It is also important to consider that T. brucei can accumulate DNA breaks, which results in parasite selection [68], while T. cruzi strongly exhibits cell cycle arrest in the presence of genotoxic agents [69].
How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase i inhibitor
2014, Molecular and Biochemical ParasitologyCitation Excerpt :Ten members of the cyclin-dependent kinase (CDK) family (as cyclin related kinase – CRK) and ten orthologous cyclins (as CYCs) were identified in the T. cruzi genome [8]. TcCYC2 was able to rescue the cell cycle in G1-arrested yeast cells [9], and some evidence suggests that CRK3 has a role in cell cycle control [10]. DNA topoisomerases play an essential role in nuclear and kDNA organization and regulate the topological state of DNA by introducing or removing supercoiling in these molecules during replication, transcription and repair [11].
Functional characterization of TcCYC2 cyclin from Trypanosoma cruzi
2012, Experimental ParasitologyCitation Excerpt :On the other hand, we studied the phenotype of TcCYC2-HA over-expressing cells synchronizing epimastigotes with hydroxyurea. The typical behavior of a synchronized T. cruzi culture follows a general rule and is reproducible within the same strain (Galanti et al., 1994; Santori et al., 2002). In our case, synchronized epimastigotes pass through all phases of the cell cycle starting from G1 upon HU release.
Regulation of a Myb transcription factor by cyclin-dependent kinase 2 in Giardia lamblia
2012, Journal of Biological ChemistryCitation Excerpt :The Cdk protein family is a group of Ser/Thr protein kinases that regulate cell cycle progression, cell proliferation, and cell differentiation in yeast, animals, and plants (50–52, 76). It has also been identified in protozoan parasites, including Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, Leishmania donovani, Leishmania major, and E. histolytica (79–84). Four Giardia Cdk homologues have been reported (84).
Nuclear Structure of Trypanosoma cruzi
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Cytokinesis in trypanosomatids
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Present Address: Molecular and Cell Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Rd, La Jolla, CA 92037, USA.