Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombe cdc2 mutant

Wang, Yixin; Dimitrov, Krassen; Garrity, Lynne K.; Sazer, Shelley; Beverley, Stephen M.

doi:10.1016/s0166-6851(98)00121-2

Cited by 33 publications

(27 citation statements)

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“…Homologues of CRK3 have been also identified and isolated from T. cruzi (TzCRK3) (27), L. mexicana (LmmCRK3) (31), and Leishmania major (LmajCRK3) (32), sharing over 75% sequence identities among themselves but only around 50% identity to human CDK1. LmajCRK3 was capable of complementing the Schizosaccharomyces pombe cdc2-33 ts mutant, demonstrating that it can carry out the cdc2 function in fission yeast (32). LmmCRK3 has been also shown as an essential gene in L. mexicana (33).…”

Section: Discussionmentioning

confidence: 99%

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

Wang

2004

Journal of Biological Chemistry

107

183

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Cyclin-dependent protein kinases are among the key regulators of eukaryotic cell cycle progression. Potential functions of the five cdc2-related kinases (CRK) in Trypanosoma brucei were analyzed using the RNA interference (RNA i ) technique. In both the procyclic and bloodstream forms of T. brucei, CRK1 is apparently involved in controlling the G 1 /S transition, whereas CRK3 plays an important role in catalyzing cells across the G 2 /M junction. A knockdown of CRK1 caused accumulation of cells in the G 1 phase without apparent phenotypic change, whereas depletion of CRK3 enriched cells of both forms in the G 2 /M phase. However, two distinctive phenotypes were observed between the CRK3-deficient procyclic and bloodstream forms. The procyclic form has a majority of the cells containing a single enlarged nucleus plus one kinetoplast. There is also an enhanced population of anucleated cells, each containing a single kinetoplast known as the zoids (0N1K). The CRK3-depleted bloodstream form has an increased number of one nucleus-two kinetoplast cells (1N2K) and a small population containing aggregated multiple nuclei and multiple kinetoplasts. Apparently, these two forms have different mechanisms in cell cycle regulation. Although the procyclic form can be driven into cytokinesis and cell division by kinetoplast segregation without a completed mitosis, the bloodstream form cannot enter cytokinesis under the same condition. Instead, it keeps going through another G 1 phase and enters a new S phase resulting in an aggregate of multiple nuclei and multiple kinetoplasts in an undivided cell. The different leakiness in cell cycle regulation between two stagespecific forms of an organism provides an interesting and useful model for further understanding the evolution of cell cycle control among the eukaryotes.

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Section: Discussionmentioning

confidence: 99%

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

Wang

2004

Journal of Biological Chemistry

107

183

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“…Homologues of CRK3 have also been identified and isolated from T. cruzi (TzCRK3) (4), Leishmania mexicana (LmmCRK3) (6), and Leishmania major (LmajCRK3) (30), sharing over 75% sequence identities. But CRK3 has only around 50% identity to human CDK1.…”

Section: Discussionmentioning

confidence: 99%

“…But CRK3 has only around 50% identity to human CDK1. LmmCRK3 is an essential gene in L. mexicana (10) capable of complementing a Schizosaccharomyces pombe cdc2 -33 ts mutant, demonstrating that it can carry out the cdc2 function in fission yeast (30). Using immunoprecipitation and a yeast two-hybrid screen, CRK3 from T. brucei was found associated with a mitotic cyclin homologue, CycB2/ CYC6, which was found indispensable for controlling G 2 /M passage in both forms of T. brucei (9,28).…”

Section: Discussionmentioning

confidence: 99%

Pairwise Knockdowns of cdc2-Related Kinases (CRKs) in Trypanosoma brucei Identified the CRKs for G ₁ /S and G ₂ /M Transitions and Demonstrated Distinctive Cytokinetic Regulations between Two Developmental Stages of the Organism

Wang

2005

Eukaryot Cell

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“…Attempts to generate a null mutant resulted in a dramatic change in the parasite's ploidy to avoid loss of this essential gene (16); this phenotype is widely interpreted to mean that the gene is essential to the organism (3, 16). CRK3 from Leishmania major complemented an S. pombe cdc2 temperature-sensitive mutant (40). CRK3 histone H1 kinase activity was inhibited in vitro with a CDK-specific inhibitor, flavopiridol, and treatment of the parasite with flavopiridol resulted in cell cycle arrest in the G 2 /M phase of the cell cycle (16).…”

mentioning

confidence: 99%

Inhibitors of Leishmania mexicana CRK3 Cyclin-Dependent Kinase: Chemical Library Screen and Antileishmanial Activity

Grant

Dunion

Yardley

et al. 2004

Antimicrob Agents Chemother

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The CRK3 cyclin-dependent kinase of Leishmania has been shown by genetic manipulation of the parasite to be essential for proliferation. We present data which demonstrate that chemical inhibition of CRK3 impairs the parasite's viability within macrophages, thus further validating CRK3 as a potential drug target. A microtiter plate-based histone H1 kinase assay was developed to screen CRK3 against a chemical library enriched for protein kinase inhibitors. Twenty-seven potent CRK3 inhibitors were discovered and screened against Leishmania donovani amastigotes in vitro. Sixteen of the CRK3 inhibitors displayed antileishmanial activity, with a 50% effective dose (ED 50 ) of less than 10 M. These compounds fell into four chemical classes: the 2,6,9-trisubstituted purines, including the C-2-alkynylated purines; the indirubins; the paullones; and derivatives of the nonspecific kinase inhibitor staurosporine. The paullones and staurosporine derivatives were toxic to macrophages. The 2,6,9-trisubstituted purines inhibited CRK3 in vitro, with 50% inhibitory concentrations ranging from high nanomolar to low micromolar concentrations. The most potent inhibitors of CRK3 (compounds 98/516 and 97/344) belonged to the indirubin class; the 50% inhibitory concentrations for these inhibitors were 16 and 47 nM, respectively, and the ED 50 s for these inhibitors were 5.8 and 7.6 M, respectively. In culture, the indirubins caused growth arrest, a change in DNA content, and aberrant cell types, all consistent with the intracellular inhibition of a cyclin-dependent kinase and disruption of cell cycle control. Thus, use of chemical inhibitors supports genetic studies to confirm CRK3 as a validated drug target in Leishmania and provides pharmacophores for further drug development.

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Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombe cdc2 mutant

Cited by 33 publications

References 50 publications

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

Pairwise Knockdowns of cdc2-Related Kinases (CRKs) in Trypanosoma brucei Identified the CRKs for G ₁ /S and G ₂ /M Transitions and Demonstrated Distinctive Cytokinetic Regulations between Two Developmental Stages of the Organism

Inhibitors of Leishmania mexicana CRK3 Cyclin-Dependent Kinase: Chemical Library Screen and Antileishmanial Activity

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Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombe cdc2 mutant

Cited by 33 publications

References 50 publications

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

Pairwise Knockdowns of cdc2-Related Kinases (CRKs) in Trypanosoma brucei Identified the CRKs for G 1 /S and G 2 /M Transitions and Demonstrated Distinctive Cytokinetic Regulations between Two Developmental Stages of the Organism

Inhibitors of Leishmania mexicana CRK3 Cyclin-Dependent Kinase: Chemical Library Screen and Antileishmanial Activity

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Pairwise Knockdowns of cdc2-Related Kinases (CRKs) in Trypanosoma brucei Identified the CRKs for G ₁ /S and G ₂ /M Transitions and Demonstrated Distinctive Cytokinetic Regulations between Two Developmental Stages of the Organism