Biochemical and enzymatic characterization of a partially purified casein kinase-1 like activity from Trypanosoma cruzi

Calabokis, Maritza; Kurz, Liliana; Wilkesman, Jeff; Galán-Caridad, José M.; Möller, Carolina; Gonzatti, Mary Isabel; Bubis, José

doi:10.1016/s1383-5769(01)00104-0

Cited by 14 publications

(15 citation statements)

References 42 publications

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“…Accordingly, these peaks were named Q-I and Q-II ( Figure 1A). These results were similar to our previous findings using T. cruzi epimastigotes that were harvested at the stationary phase of growth [5]. Two synthetic peptides, P1 which is specific for CK1, and P2 which is specifically recognized by CK2, were examined as substrates for Q-I and Q-II.…”

Section: Purification Of a T Cruzi Ck1 Activitysupporting

confidence: 90%

“…Yet, database analysis of the complement of protein kinases in the T. cruzi genome identified seven putative CK1 protein kinase genes [8]. Here, we have purified a CK1 activity from the exponential phase of T. cruzi epimastigotes, which functionally differs from the parasite CK1 enzymes previously characterized by Calabokis et al, [5,6] and Spadafora et al, [7], and might correspond to a mixed kinase activity. L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), phenyl methyl sulfonyl fluoride (PMSF), heparin, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), emodin, ATP, GTP, 5'-guanylylimidodiphosphate (GMP-PNP), Q-sepharose, Sigma; N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7), 1-(8-chloro-5-isoquinolinesulfonyl)piperazine (CKI-8), Seikagaku America; P81 phosphocellulose chromatography paper, Whatman; OptiPhase Hisafe II (liquid scintillation counting solution), LKB; bicinchoninic acid BCA TM Protein Assay Kit, Pierce.…”

Section: Introductionmentioning

confidence: 83%

“…CK1 is evolutionary conserved within eukaryotes and regulates a plethora of cellular processes [1][2][3][4]. We have purified and characterized a CK1 enzyme from the stationary phase of growth of T. cruzi epimastigotes [5,6]. Additionally, Spadafora et al, [7] have cloned two CK1 isoforms from T. cruzi, TcCK1.1 and TcCK1.2, which appeared to be differentially expressed throughout the parasite life cycle by Northern blot analyses.…”

Section: Introductionmentioning

confidence: 99%

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An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

Justiniano¹,

Noris-Suárez²,

Lima³

et al. 2014

Bio Chem Comp

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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 -32 P] 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 K m 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.

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Section: Purification Of a T Cruzi Ck1 Activitysupporting

confidence: 90%

Section: Introductionmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

Justiniano¹,

Noris-Suárez²,

Lima³

et al. 2014

Bio Chem Comp

Self Cite

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show abstract

“…Calabokis et al (2002) have reported a soluble CK1-like activity in T. cruzi, which showed similar biochemical properties to other CK1s from higher eukaryotes. The importance of CK1 for the control of some essential cellular functions was demonstrated by genetic studies in yeast (Robinson et al 1992).…”

Section: Discussionmentioning

confidence: 53%

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Galán-Caridad

Calabokis

Uzcanga

et al. 2004

Mem. Inst. Oswaldo Cruz

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“…Protein kinase assays were performed according to Calabokis et al (2002). The reaction mixtures were carried out in a final volume of 70 µl and contained 50 mM Tris-HCl (pH 8.0), 12 mM MgCl 2 , 20 mM KF, 100 µM [γ 32 P] ATP (specific activity ≈ 4500 cpm/pmol), and 50 µl of the parasite fraction.…”

Section: Protein Kinase Assaymentioning

confidence: 99%