Two casein kinase 1 isoforms are differentially expressed in Trypanosoma cruzi

Spadafora, Carmenza; Repetto, Yolanda; Torres, Cristina; Pino, Laura; Robello, Carlos; Morello, Antonio; Gamarro, Francisco; Castanys, Santiago

doi:10.1016/s0166-6851(02)00156-1

Cited by 16 publications

(20 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The enzyme was adsorbed to the resin when the Q-sepharose flow-through fraction containing the activated casein kinase activity was applied to a second Q-sepharose column, and then, the enzyme was purified after the appropriate elution by increasing the ionic strength of the buffer. On the basis of kinetic measurements, this enzyme was different from the CK1 enzymes previously characterized from T. cruzi [5,7]. This novel enzyme has K m values of 172.5±5.1 µM, 0.2062±0.0051 mg/ml and 35.5±2.9 μM for ATP, casein and P1, respectively, which varied from the values previously reported for the purified CK1 enzyme from the stationary phase of growth of T. cruzi epimastigotes [5], and for the recombinant TcCK1.1 isoform [7], which appeared to represent the same CK1 enzyme (product of the Tc00.1047053508541.220 gene).…”

Section: Discussionmentioning

confidence: 73%

“…On the basis of kinetic measurements, this enzyme was different from the CK1 enzymes previously characterized from T. cruzi [5,7]. This novel enzyme has K m values of 172.5±5.1 µM, 0.2062±0.0051 mg/ml and 35.5±2.9 μM for ATP, casein and P1, respectively, which varied from the values previously reported for the purified CK1 enzyme from the stationary phase of growth of T. cruzi epimastigotes [5], and for the recombinant TcCK1.1 isoform [7], which appeared to represent the same CK1 enzyme (product of the Tc00.1047053508541.220 gene). Given that epimastigotes were collected here at the exponential phase of growth, whereas parasites were collected during the stationary phase of growth in the study reported by Calabokis et al, [5], the enzyme purified in this manuscript must correspond to another one of the seven different CK1 isoforms that are present in T. cruzi [8].…”

Section: Discussionmentioning

confidence: 73%

“…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: 82%

“…In a second differentiation process, known as metacyclogenesis, which occurs within the vector hindgut, epimastigotes are transformed to non-proliferative, infectious, metacyclic trypomastigotes. Spadafora et al, [7] analyzed the total parasite CKl activity during the cell growth of T. cruzi epimastigote forms, and reported that the CK1 activity increased in the exponential phase of growth until parasites reached the late exponential phase, at which point a drop in activity was observed. Moreover, they also showed through synchronization studies, that the CK1 activity has been most conspicuously circumscribed to the S and M phases of the cell cycle [7].…”

Section: Discussionmentioning

confidence: 99%

“…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. Yet, database analysis of the complement of protein kinases in the T. cruzi genome identified seven putative CK1 protein kinase genes [8].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

Justiniano¹,

Noris-Suárez²,

Lima³

et al. 2014

Bio Chem Comp

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

Justiniano¹,

Noris-Suárez²,

Lima³

et al. 2014

Bio Chem Comp

View full text Add to dashboard Cite

show abstract

The composition of upstream open reading frames (uORF) in four genes from Trypanosoma cruzi typical strains

Jaeger

Brandão

2011

Parasitol Res

View full text Add to dashboard Cite

Upstream open reading frames (uORF) are small open reading frames located in the 5' untranslated region (5' utr) of a mature mRNA. We analysed in four strains representing the Trypanosoma cruzi groups Tc I, Tc II, Tc IV and Tc VI the uORF present in 5' utr sequences of four genes: P-type H+-ATPase 1, DEAD/H RNA helicase, casein kinase 1.1 and ferredoxin-NADP+ reductase. A segment in the 5' utr at each of these genes encompassing one or more uORF was PCR amplified and sequenced. An analysis of these sequences reveals that the uORF in T. cruzi show minor variations; however, these nucleotide substitutions mirror the divergence of T. cruzi strains into major groups.

show abstract

Protein kinases as targets for anti-parasitic chemotherapy

Doerig

2004

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

123

View full text Add to dashboard Cite

Parasitic protozoa infecting humans have a staggering impact on public health, especially in the developing world. Furthermore, several protozoan species are major pathogens of domestic animals and have a considerable impact on food production, largely but not exclusively in developing countries. In many instances, the parasites have developed resistance against available chemotherapeutic agents, making the search for alternative drugs a priority [1]. In line with the current interest in protein kinases inhibitors as potential drugs against a variety of infectious and non-infectious diseases, the possibility that protein kinases may represent targets for novel anti-parasitic agents is being explored in a variety of protozoan parasites [2,3]. Research into parasite protein kinases has benefited greatly from genome and EST sequencing projects, with the genomes of a few species fully sequenced (notably that of the human malaria parasite Plasmodium falciparum) and several more under way. The overall picture that emerged from research in this area shows (i) that protozoan parasites, expectedly, possess homologues of many of the serine/threonine protein kinase families found in other eukaryotes (but no gene encoding tyrosine kinases have yet been identified in these organisms), and (ii) that protein kinase-based regulatory networks similar to those found in higher Eukaryotes, such as some of the major signal transduction pathways and the cell cycle machinery, operate in these organisms. However, the phylogenetic isolation of parasitic protozoa is reflected by atypical structural and functional properties of many of their protein kinase homologues. Likewise, evidence is emerging, which suggests that the organisation of some otherwise well conserved signal transduction pathways is divergent in some parasitic species. Selected examples of such divergences drawn from a variety of protozoan parasites, concerning for instance the CDKs, and the MAPK and cyclic nucleotide pathways, will be discussed. The differences between protein kinases of a parasite and their homologues in its host cell suggest that specific inhibition of the former can be achieved. The problem of the validation of these enzymes as drug targets will be discussed, with emphasis on gene knock-out and chemical genetics approaches as possible solutions. The development of anti-parasitic drugs based on protein kinase inhibition is being pursued following two avenues [2,3]: one consists of screening chemical libraries on recombinant enzymes; several protein kinases from parasitic protozoa are now available for this approach, and preliminary results will be presented. The second approach relies on the identification of the molecular targets of kinase inhibitors which have previously been shown to

show abstract

Two casein kinase 1 isoforms are differentially expressed in Trypanosoma cruzi

Cited by 16 publications

References 50 publications

An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

The composition of upstream open reading frames (uORF) in four genes from Trypanosoma cruzi typical strains

Protein kinases as targets for anti-parasitic chemotherapy

Contact Info

Product

Resources

About