Biochemical Compounds

Biochemical Compounds

ISSN 2052-9341
Original Research

An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

Irene Justiniano1, Karem Noris-Suarez2, Ana R. De Lima3, Victor T. Contreras3 and José Bubis2*

*Correspondence: José Bubis

2. Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela.

Author Affiliations

1. Departamento de Química, Universidad Simón Bolívar, Caracas, Venezuela.

3. Laboratorio de Protozoología, Centro BioMolP, Facultad de Ciencias de la Salud, Universidad de Carabobo, Valencia, Venezuela.


Background: CK1 enzymes are serine/threonine protein kinases that regulate numerous cellular processes. Although seven putative CK1 ortholog genes have been identified in the genome of Trypanosoma cruzi, the causative agent of Chagas' disease, only two parasite CK1 isoforms have been characterized to date.

Methods: T. cruzi epimastigotes were collected at the exponential phase of growth. The parasite clarified cytosolic fraction was separated by anion-exchange column chromatography, and the non-adsorbed fraction was rechromatographed on a second anion-exchange column. In order to identify the parasite casein kinases, ATP:phosphotransferase activity was evaluated by using dephosphorylated casein as substrate and [γ-32P] ATP as cosubstrate. In addition, specific peptide substrates and inhibitors for higher eukaryotic CK1 and CK2 enzymes were employed to classify the purified T. cruzi protein kinase.

Results: A soluble protein kinase that uses casein as a substrate, and possesses an apparent molecular weight of 33,000, was purified from the exponential phase of growth of T. cruzi epimastigotes. The purified protein specifically phosphorylated P1 (sequence=RRKDLHDDEEDEAMSITA), a selective peptide substrate for CK1, but not P2 (sequence=RRRADDSDDDDD) which is a CK2 substrate, and was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide and 1-(8-chloro-5-isoquinolinesulfonyl)piperazine, two specific inactivators of CK1. Consequently, the purified casein kinase was classified as a CK1 enzyme. Kinetic studies showed that the T. cruzi CK1 has a Km of 172.5±5.1 µM, 0.2062±0.0051 mg/ml and 35.5±2.9 µM for ATP, casein and P1, respectively. Interestingly, this CK1 was stimulated in the presence of about 0.1 mM GTP or 5'-guanylylimidodiphosphate, but was inhibited at approximately 1 mM of either of these guanine nucleotides. Additionally, this enzyme was more than 80% inactivated by low concentrations of heparin (1 µM), which is a common inhibitor of CK2 enzymes. However, other inhibitors of mammalian CK2, such as emodin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, only affected the parasite CK1 activity at the highest concentrations used. Our findings demonstrate that this CK1 from T. cruzi has a dual modulation by GTP and an unusual sensitivity for heparin.

Conclusions: A novel CK1 activity that functionally differs from previously characterized T. cruzi CK1 enzymes was purified from the exponential phase of growth of epimastigote forms.

Keywords: Trypanosoma cruzi, epimastigotes, CK1, casein kinase 1, protein kinase, signaling

ISSN 2052-9341
Volume 2
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