Evidence for CRK3 participation in the cell division cycle of Trypanosoma cruzi

https://doi.org/10.1016/S0166-6851(02)00039-7Get rights and content

Abstract

Trypanosoma cruzi CRK3 gene encodes a Cdc2p related protein kinase (CRK). To establish if it has a role in the regulation of the parasite cell cycle we studied CRK3 expression and activity throughout three life cycle stages. CRK3 from epimastigote soluble extracts interacted with p13suc1-beads. Endogenous CRK3 phosphorylated histone H1 and this activity was inhibited by specific CDK inhibitors: Olomoucine, Flavopiridol and Roscovitine. Flavopiridol partially inhibited the growth of T. cruzi epimastigotes at 50 nM, the lowest concentration used, but even with the highest (5 μM), cell growth was not completely arrested. CRK3 from Flavopiridol-inhibited epimastigote extracts exhibited a dose dependent inhibition of histone H1 phosphorylation. T. cruzi p13suc1-binding CRK displayed the same inhibition profile. This suggests that CRK3 is the enzyme responsible for the majority of the kinase activity associated with p13suc1. CRK3 activity of hydroxyurea (HU) synchronized epimastigotes peaked in G2/M boundary while the kinase activity associated to p13suc1-beads increased at the same time point but remained high until late G2/M. In addition, CRK3 expression was constant during the cell cycle. This is a common pattern of CDK activity regulation. Taken together, these results support the idea that CRK3 is involved in control of the cell cycle in T. 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;

References (35)

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Present Address: Molecular and Cell Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Rd, La Jolla, CA 92037, USA.

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