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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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Related in: MedlinePlus

Effects of mammalian PKA inhibitors on the kinase activity of TgPKA-C in vitro.A and B, Kinase activities of TgPKA-C and BtPKA-C were calculated in the presence of 400, 200, 100, 50 and 25 µM of H89 (A) or 100 µM of bovine PKI (B). Reactions were carried out at 30°C for 30 min and terminated by boiling in SDS-PAGE sample buffer. Reaction mixtures were then subjected to resolution by 15% SDS-PAGE followed by Coomassie staining. Phosphate incorporation into Histone IIAS was measured by scintillation counting of excised gel fragments. Enzyme activity in the absence of inhibitors was taken to be 100%. Representative results of three independent determinations are shown.
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pone-0022492-g004: Effects of mammalian PKA inhibitors on the kinase activity of TgPKA-C in vitro.A and B, Kinase activities of TgPKA-C and BtPKA-C were calculated in the presence of 400, 200, 100, 50 and 25 µM of H89 (A) or 100 µM of bovine PKI (B). Reactions were carried out at 30°C for 30 min and terminated by boiling in SDS-PAGE sample buffer. Reaction mixtures were then subjected to resolution by 15% SDS-PAGE followed by Coomassie staining. Phosphate incorporation into Histone IIAS was measured by scintillation counting of excised gel fragments. Enzyme activity in the absence of inhibitors was taken to be 100%. Representative results of three independent determinations are shown.

Mentions: An inhibition assay was used to estimate the effect of PKA specific inhibitors on TgPKA-C. As shown in Figure 4A, inhibition assays of TgPKA-C by H89 was performed on the concentrations of 400, 200, 100, 50 and 25 µM. The estimated IC50 was 175 µM, which might be much higher than that of mammal PKA [31]. Inhibitory assay of PKI was performed in the presence or absence of 100 µM PKI5-24. In addition to TgPKA-C, Bos taurus PKA-C (BtPKA-C; Promega, Madison, WI) was included as a positive control. Compared to BtPKA-C, inhibition by PKI was much weaker in TgPKA-C (Figure 4B). These data indicate that susceptibility of TgPKA-C on both inhibitors differs from that of mammalian PKA-C.


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)

Effects of mammalian PKA inhibitors on the kinase activity of TgPKA-C in vitro.A and B, Kinase activities of TgPKA-C and BtPKA-C were calculated in the presence of 400, 200, 100, 50 and 25 µM of H89 (A) or 100 µM of bovine PKI (B). Reactions were carried out at 30°C for 30 min and terminated by boiling in SDS-PAGE sample buffer. Reaction mixtures were then subjected to resolution by 15% SDS-PAGE followed by Coomassie staining. Phosphate incorporation into Histone IIAS was measured by scintillation counting of excised gel fragments. Enzyme activity in the absence of inhibitors was taken to be 100%. Representative results of three independent determinations are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022492-g004: Effects of mammalian PKA inhibitors on the kinase activity of TgPKA-C in vitro.A and B, Kinase activities of TgPKA-C and BtPKA-C were calculated in the presence of 400, 200, 100, 50 and 25 µM of H89 (A) or 100 µM of bovine PKI (B). Reactions were carried out at 30°C for 30 min and terminated by boiling in SDS-PAGE sample buffer. Reaction mixtures were then subjected to resolution by 15% SDS-PAGE followed by Coomassie staining. Phosphate incorporation into Histone IIAS was measured by scintillation counting of excised gel fragments. Enzyme activity in the absence of inhibitors was taken to be 100%. Representative results of three independent determinations are shown.
Mentions: An inhibition assay was used to estimate the effect of PKA specific inhibitors on TgPKA-C. As shown in Figure 4A, inhibition assays of TgPKA-C by H89 was performed on the concentrations of 400, 200, 100, 50 and 25 µM. The estimated IC50 was 175 µM, which might be much higher than that of mammal PKA [31]. Inhibitory assay of PKI was performed in the presence or absence of 100 µM PKI5-24. In addition to TgPKA-C, Bos taurus PKA-C (BtPKA-C; Promega, Madison, WI) was included as a positive control. Compared to BtPKA-C, inhibition by PKI was much weaker in TgPKA-C (Figure 4B). These data indicate that susceptibility of TgPKA-C on both inhibitors differs from that of mammalian PKA-C.

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
Related in: MedlinePlus