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

Galán-Caridad, José M.; Calabokis, Maritza; Uzcanga, Graciela L.; Aponte, Frank; Bubis, José

doi:10.1590/s0074-02762004000800011

Cited by 10 publications

(2 citation statements)

References 37 publications

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“…Interestingly, when the purified parasite CK1 activity was examined in the presence of GTP, we initially observed an unusual activation on the enzymatic activity at concentrations of about 100 μM of GTP, which was followed by an inhibition of its enzymatic activity at higher concentrations of GTP. A similar effect was reported for a casein kinase-like activity in crude extracts and particulate fractions of the Teva1 isolate of Trypanosoma evansi, but not in its soluble fractions [18]. This effect was proposed to be caused by either a GTP-activated protein kinase or a GTP-dependent inactivation of a phosphatase.…”

Section: Discussionsupporting

confidence: 70%

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: Discussionsupporting

confidence: 70%

An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

Justiniano¹,

Noris-Suárez²,

Lima³

et al. 2014

Bio Chem Comp

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“…Evidence of PKA activity has also been uncovered in Trypanosoma evansi . Intriguingly, cAMP also had no effect on the phosphorylating activity of whole‐cell lysates or particulate fractions, but a doubling of kinase activity was recorded on the addition of cAMP in the soluble fraction (Galan‐Caridad et al ., 2004). This stimulation could be inhibited almost completely by the addition of PKI.…”

Section: Kinetoplastid Pkamentioning

confidence: 99%

Cyclic-nucleotide signalling in protozoa

Gould

Koning

2011

FEMS Microbiol Rev

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Compared with the impressive progress in understanding signal transduction pathways and mechanisms in mammalian systems, advances in protozoan signalling processes, including cyclic nucleotide metabolism, have been very slow. This is in large part connected to the fact that the components of these pathways are very different in the protozoan parasites, as confirmed by the recently completed genome. For instance, kinetoplastids have no equivalents to the mammalian Class I adenylyl cyclases (ACs) in their genomes nor any of the subunits of the associated G-proteins. The cyclases in kinetoplastid parasites contain a single transmembrane domain, a conserved intracellular catalytic domain and a highly variable extracellular domain - consistent with the expression of multiple receptor-activated cyclases - but no receptor ligands, agonists or antagonists have been identified. Apicomplexan AC and guanylyl cyclase (GC) are even more unusual, potentially being bifunctional, harbouring either a putative ion channel (AC) or a P-type ATPase-like domain (GC) alongside the catalytic region. Phosphodiesterases (PDEs) and cyclic-nucleotide-activated protein kinases are essentially conserved in protozoa, although mostly insensitive to inhibitors of the mammalian proteins. Some of the PDEs have now been validated as promising drug targets. In the following manuscript, we will summarize the existing literature on cAMP and cGMP in protozoa: cyclases, PDEs and cyclic-nucleotide-dependent kinases.

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