• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • CK enzymes belong to a conserved family of serine threonine


    CK1 enzymes belong to a conserved family of serine/threonine protein kinases that plays an important and diverse role in vesicular trafficking, DNA repair, CHIR-124 progression and cytokinesis in organisms from yeast to humans [15]. In multicellular organisms CK1 enzymes also regulate developmental pathways, control circadian rhythms and have been implicated in Alzheimer\'s disease progression [16]. S. cerevisiae encodes four CK1 isoforms, Yck1p, Yck2p, Yck3p and Hrr25p, which constitute two sets of functionally redundant gene pairs (Yck1p/Yck2p and Yck3p/Hrr25p) that are essential for growth [17], [18], [19]. A CK1 gene also appears to be essential for vegetative growth in Dictyostelium[20]. CK1 enzymes have been described for Plasmodium, Leishmania and Trypanosma parasites [21], [22], [23], [24], [25], but genetic or pharmacological evidence of their significance is currently lacking. Here we report the cloning, expression and characterization of two CK1 isoforms of T. gondii and describe their sensitivity to a collection of structurally diverse CDK inhibitors. As predicted from earlier affinity-ligand purification studies [11], we have confirmed the sensitivity of one of these isoforms to the tri-substituted purine compound purvalanol B. We show that aminopurvalanol, a structurally related compound and more potent TgCK1α inhibitor, also inhibits T. gondii growth. These results encourage further investigation of parasite TgCK1α as a potential chemotherapeutic target.
    Materials and methods
    Discussion In this report we provide evidence that supports and extends the results of a previous study that implicated parasite CK1 as the intracellular target of a trisubstituted purine CDK inhibitor purvalanol B [11]. Using purvalanol B as an affinity ligand, CK1 enzymes were captured from lysates prepared from a variety of apicomplexan (P. falciparum and T. gondii) and kinetoplastid (T. cruzi and L. mexicana) parasites. Since active enzymes were not recovered from the affinity matrix in these experiments, an evaluation of the selectivity of purvalanol B and related compounds for their putative target has awaited the cloning, expression and characterization of parasite CK1 enzymes. We have now cloned the products of two different Toxoplasma loci that encode α and β CK1 isoforms and have transiently expressed the active recombinant enzymes in tachyzoites as well as in E. coli. The TgCK1α isoform matches the identity of the CK1 protein associated with the purvalanol B affinity matrix and the corresponding native enzyme was partially purified from tachyzoites. In contrast, it was not possible to detect a protein corresponding to the native TgCK1β isoform in fractionated detergent extracts of tachyzoites with a variety of affinity-purified sera raised CHIR-124 against TgCK1 peptide epitopes (Fig. 1, Fig. 4, Fig. 5). The absence of a second kinase isoform in tachyzoites despite the presence of a cognate transcript has also been observed for TgCDPK isoform 2 (TgCDPK2) and suggests that expression of different kinase isoforms might be restricted to a particular developmental stage [31]. Recombinant TgCK1β expressed transiently in tachyzoites was associated with surface membranes and the determinant responsible for this association mapped to a C-terminal extension that is absent from isoform α. Unlike the catalytic core domain, the C-terminal domains of CK1 enzymes are commonly quite variable in plants and in animals and are thought to confer on the enzyme unique properties of regulatory responsiveness, intracellular location, or tissue specificity [15], [36]. In the case of the S. cerevisiae CK1 genes YCK1 and YCK2, the corresponding C-terminal region mediates membrane association through acylation of a terminal pair of cysteine residues, although proximal sequences are also required for modification and targeting [18], [52], [53]. We have not identified the precise nature of the determinant conferring membrane association on TgCK1β, but obvious features such as a transmembrane domain or consensus sites for C-terminal acylation appear to be absent.