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Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth.

Kurokawa H, Kato K, Iwanaga T, Sugi T, Sudo A, Kobayashi K, Gong H, Takemae H, Recuenco FC, Horimoto T, Akashi H - PLoS ONE (2011)

Bottom Line: The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated.Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT

Background: cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/principal finding: The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/significance: Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

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Interaction of GST-TgPKA-C and MBP-TgPKA-R.A, Purified MBP-β-gal (lane 1) or MBP-TgPKA-R (lane 2) was incubated with GST-TgPKA-C in kinase buffer containing [γ-32P]ATP, separated on a denaturing gel, and Coomassie stained. B, Autoradiograph of the gel shown in (A). Arrows indicate the migration of GST-TgPKA-C, MBP-β-gal, or MBP-TgPKA-R. Molecular masses (kDa) are shown on the left.
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pone-0022492-g006: Interaction of GST-TgPKA-C and MBP-TgPKA-R.A, Purified MBP-β-gal (lane 1) or MBP-TgPKA-R (lane 2) was incubated with GST-TgPKA-C in kinase buffer containing [γ-32P]ATP, separated on a denaturing gel, and Coomassie stained. B, Autoradiograph of the gel shown in (A). Arrows indicate the migration of GST-TgPKA-C, MBP-β-gal, or MBP-TgPKA-R. Molecular masses (kDa) are shown on the left.

Mentions: To determine whether TgPKA-R interacts with TgPKA-C in vitro, we used TgPKA-C and recombinant TgPKA-R in an in vitro kinase assay. We first expressed TgPKA-R as an MBP fusion protein. Next, we incubated purified MBP-TgPKA-R with GST-TgPKA-C. MBP-β-gal-α was used as a negative control. As shown in Figure 6A, lane 1, MBP-β-gal-α did not show evidence of a positive reaction. However, in the autoradiographic image of purified MBP-TgPKA-R, a protein band with an apparent Mr of 86,000 was labeled (Figure 6B, lane 2). These data demonstrate that TgPKA-C phosphorylates TgPKA-R in vitro.


Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth.

Kurokawa H, Kato K, Iwanaga T, Sugi T, Sudo A, Kobayashi K, Gong H, Takemae H, Recuenco FC, Horimoto T, Akashi H - PLoS ONE (2011)

Interaction of GST-TgPKA-C and MBP-TgPKA-R.A, Purified MBP-β-gal (lane 1) or MBP-TgPKA-R (lane 2) was incubated with GST-TgPKA-C in kinase buffer containing [γ-32P]ATP, separated on a denaturing gel, and Coomassie stained. B, Autoradiograph of the gel shown in (A). Arrows indicate the migration of GST-TgPKA-C, MBP-β-gal, or MBP-TgPKA-R. Molecular masses (kDa) are shown on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140512&req=5

pone-0022492-g006: Interaction of GST-TgPKA-C and MBP-TgPKA-R.A, Purified MBP-β-gal (lane 1) or MBP-TgPKA-R (lane 2) was incubated with GST-TgPKA-C in kinase buffer containing [γ-32P]ATP, separated on a denaturing gel, and Coomassie stained. B, Autoradiograph of the gel shown in (A). Arrows indicate the migration of GST-TgPKA-C, MBP-β-gal, or MBP-TgPKA-R. Molecular masses (kDa) are shown on the left.
Mentions: To determine whether TgPKA-R interacts with TgPKA-C in vitro, we used TgPKA-C and recombinant TgPKA-R in an in vitro kinase assay. We first expressed TgPKA-R as an MBP fusion protein. Next, we incubated purified MBP-TgPKA-R with GST-TgPKA-C. MBP-β-gal-α was used as a negative control. As shown in Figure 6A, lane 1, MBP-β-gal-α did not show evidence of a positive reaction. However, in the autoradiographic image of purified MBP-TgPKA-R, a protein band with an apparent Mr of 86,000 was labeled (Figure 6B, lane 2). These data demonstrate that TgPKA-C phosphorylates TgPKA-R in vitro.

Bottom Line: The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated.Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT

Background: cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/principal finding: The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/significance: Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

Show MeSH