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Nuclear DBF-2-related kinases are essential regulators of cytokinesis in bloodstream stage Trypanosoma brucei.

Ma J, Benz C, Grimaldi R, Stockdale C, Wyatt P, Frearson J, Hammarton TC - J. Biol. Chem. (2010)

Bottom Line: Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited.Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo.Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection & Immunity, Faculty of Biomedical and Life Sciences and Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.

ABSTRACT
Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

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PK50 and PK53 are essential for proliferation in bloodstream stage T. brucei. A, cumulative growth curves for two independent PK50 (left) and PK53 (right) bloodstream stage RNAi clones in the presence and absence of tetracycline (tet). B, real time PCR, 10 h post-induction for PK50 (left) and PK53 (right) RNAi clones with PK50- and PK53-specific oligonucleotides (oligos). RT, reverse transcriptase. Error bars show the standard error over three replicates. C, Western blotting, 15 h post-induction for PK50 (left) and PK53 (right) RNAi clones with anti-PK50 (top panel) and anti-PK53 (middle panel) antibodies. The lower panel shows part of the polyvinylidene difluoride membrane following blotting, stained with Ponceau S, as a loading control. 106 cell eq were loaded per lane.
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Figure 1: PK50 and PK53 are essential for proliferation in bloodstream stage T. brucei. A, cumulative growth curves for two independent PK50 (left) and PK53 (right) bloodstream stage RNAi clones in the presence and absence of tetracycline (tet). B, real time PCR, 10 h post-induction for PK50 (left) and PK53 (right) RNAi clones with PK50- and PK53-specific oligonucleotides (oligos). RT, reverse transcriptase. Error bars show the standard error over three replicates. C, Western blotting, 15 h post-induction for PK50 (left) and PK53 (right) RNAi clones with anti-PK50 (top panel) and anti-PK53 (middle panel) antibodies. The lower panel shows part of the polyvinylidene difluoride membrane following blotting, stained with Ponceau S, as a loading control. 106 cell eq were loaded per lane.

Mentions: Purified GST:PK50 and GST:PK53 were sent to the Scottish Antibody Production Unit, Penicuik, Scotland, United Kingdom, for immunization of sheep to generate specific antisera against PK50 and PK53, which were affinity-purified using protein A/G and GST columns sequentially and stored in phosphate-buffered saline. Western blotting showed the purified anti-PK50 and anti-PK53 antiserum (used at 1:100 dilution) to recognize proteins of the predicted sizes for PK50 (50.1 kDa) and PK53 (53.0 kDa) in trypanosome cell lysates that were depleted following PK50 or PK53 RNAi (Fig. 1C).


Nuclear DBF-2-related kinases are essential regulators of cytokinesis in bloodstream stage Trypanosoma brucei.

Ma J, Benz C, Grimaldi R, Stockdale C, Wyatt P, Frearson J, Hammarton TC - J. Biol. Chem. (2010)

PK50 and PK53 are essential for proliferation in bloodstream stage T. brucei. A, cumulative growth curves for two independent PK50 (left) and PK53 (right) bloodstream stage RNAi clones in the presence and absence of tetracycline (tet). B, real time PCR, 10 h post-induction for PK50 (left) and PK53 (right) RNAi clones with PK50- and PK53-specific oligonucleotides (oligos). RT, reverse transcriptase. Error bars show the standard error over three replicates. C, Western blotting, 15 h post-induction for PK50 (left) and PK53 (right) RNAi clones with anti-PK50 (top panel) and anti-PK53 (middle panel) antibodies. The lower panel shows part of the polyvinylidene difluoride membrane following blotting, stained with Ponceau S, as a loading control. 106 cell eq were loaded per lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2865264&req=5

Figure 1: PK50 and PK53 are essential for proliferation in bloodstream stage T. brucei. A, cumulative growth curves for two independent PK50 (left) and PK53 (right) bloodstream stage RNAi clones in the presence and absence of tetracycline (tet). B, real time PCR, 10 h post-induction for PK50 (left) and PK53 (right) RNAi clones with PK50- and PK53-specific oligonucleotides (oligos). RT, reverse transcriptase. Error bars show the standard error over three replicates. C, Western blotting, 15 h post-induction for PK50 (left) and PK53 (right) RNAi clones with anti-PK50 (top panel) and anti-PK53 (middle panel) antibodies. The lower panel shows part of the polyvinylidene difluoride membrane following blotting, stained with Ponceau S, as a loading control. 106 cell eq were loaded per lane.
Mentions: Purified GST:PK50 and GST:PK53 were sent to the Scottish Antibody Production Unit, Penicuik, Scotland, United Kingdom, for immunization of sheep to generate specific antisera against PK50 and PK53, which were affinity-purified using protein A/G and GST columns sequentially and stored in phosphate-buffered saline. Western blotting showed the purified anti-PK50 and anti-PK53 antiserum (used at 1:100 dilution) to recognize proteins of the predicted sizes for PK50 (50.1 kDa) and PK53 (53.0 kDa) in trypanosome cell lysates that were depleted following PK50 or PK53 RNAi (Fig. 1C).

Bottom Line: Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited.Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo.Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection & Immunity, Faculty of Biomedical and Life Sciences and Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.

ABSTRACT
Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

Show MeSH
Related in: MedlinePlus