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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.

Show MeSH
Expression, purification and kinase activity of TgPKA-C.A, A silver-stained ge and an immunoblot of purified GST-GFP or GST-TgPKA-C generated in the wheat germ cell-free protein synthesis system using pEU-GST-GFP (lanes 1 and 2) or pEU-GST-TgPKA-C (lanes 3 and 4). Total wheat germ extracts (lanes 1 and 3) were subjected to affinity chromatography on glutathione-Sepharose beads (lanes 2 and 4). The proteins were separated on denaturing gels and subjected to silver staining or transferred onto a nitrocellulose sheet and reacted with the anti-GST antibody. Molecular masses (kDa) are shown on the left. B, Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer containing [γ-32P]ATP, separated on a denaturing gel, and Coomassie stained (left panel). Autoradiograph of the gel (right panel). Arrows indicate the migration of GST-TgPKA-C and GST-GFP. C, Purified GST-TgPKA-C was incubated in kinase buffer (lane 1). The labeled protein was treated with λ-protein phosphatase (lane 2). Reaction mixtures were then subjected to resolution on an 8% SDS-PAGE gel, followed by Coomassie staining (left panel). Autoradiograph of the gel (C). D, Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer with Histone IIAS and separated on a 15% denaturing gel followed by Coomassie staining (left panel). Autoradiograph of the gel (right panel). Molecular masses (kDa) are shown on the left.
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pone-0022492-g003: Expression, purification and kinase activity of TgPKA-C.A, A silver-stained ge and an immunoblot of purified GST-GFP or GST-TgPKA-C generated in the wheat germ cell-free protein synthesis system using pEU-GST-GFP (lanes 1 and 2) or pEU-GST-TgPKA-C (lanes 3 and 4). Total wheat germ extracts (lanes 1 and 3) were subjected to affinity chromatography on glutathione-Sepharose beads (lanes 2 and 4). The proteins were separated on denaturing gels and subjected to silver staining or transferred onto a nitrocellulose sheet and reacted with the anti-GST antibody. Molecular masses (kDa) are shown on the left. B, Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer containing [γ-32P]ATP, separated on a denaturing gel, and Coomassie stained (left panel). Autoradiograph of the gel (right panel). Arrows indicate the migration of GST-TgPKA-C and GST-GFP. C, Purified GST-TgPKA-C was incubated in kinase buffer (lane 1). The labeled protein was treated with λ-protein phosphatase (lane 2). Reaction mixtures were then subjected to resolution on an 8% SDS-PAGE gel, followed by Coomassie staining (left panel). Autoradiograph of the gel (C). D, Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer with Histone IIAS and separated on a 15% denaturing gel followed by Coomassie staining (left panel). Autoradiograph of the gel (right panel). Molecular masses (kDa) are shown on the left.

Mentions: To characterize the function of the protein, we expressed and purified TgPKA-C as a GST fusion protein using a wheat germ cell-free protein synthesis system. Purified protein was then electrophoretically separated in a denaturing gel and either silver-stained or immunoblotted with rabbit antiserum containing α-GST antibody (Figure 3A). The purified proteins from the wheat germ extracts after cell-free protein synthesis with either pEU-GST-GFP [for the expression of GST and green fluorescence protein (GFP) fusion protein] or pEU-GST-TgPKA-C contained one major purified protein with an Mr of 53,000 or 69,000, respectively, as detected by silver staining. These proteins reacted with antiserum containing α-GST antibody. These data indicate that we successfully purified the desired GST fusion proteins.


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)

Expression, purification and kinase activity of TgPKA-C.A, A silver-stained ge and an immunoblot of purified GST-GFP or GST-TgPKA-C generated in the wheat germ cell-free protein synthesis system using pEU-GST-GFP (lanes 1 and 2) or pEU-GST-TgPKA-C (lanes 3 and 4). Total wheat germ extracts (lanes 1 and 3) were subjected to affinity chromatography on glutathione-Sepharose beads (lanes 2 and 4). The proteins were separated on denaturing gels and subjected to silver staining or transferred onto a nitrocellulose sheet and reacted with the anti-GST antibody. Molecular masses (kDa) are shown on the left. B, Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer containing [γ-32P]ATP, separated on a denaturing gel, and Coomassie stained (left panel). Autoradiograph of the gel (right panel). Arrows indicate the migration of GST-TgPKA-C and GST-GFP. C, Purified GST-TgPKA-C was incubated in kinase buffer (lane 1). The labeled protein was treated with λ-protein phosphatase (lane 2). Reaction mixtures were then subjected to resolution on an 8% SDS-PAGE gel, followed by Coomassie staining (left panel). Autoradiograph of the gel (C). D, Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer with Histone IIAS and separated on a 15% denaturing gel followed by Coomassie staining (left panel). Autoradiograph of the gel (right panel). Molecular masses (kDa) are shown on the left.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140512&req=5

pone-0022492-g003: Expression, purification and kinase activity of TgPKA-C.A, A silver-stained ge and an immunoblot of purified GST-GFP or GST-TgPKA-C generated in the wheat germ cell-free protein synthesis system using pEU-GST-GFP (lanes 1 and 2) or pEU-GST-TgPKA-C (lanes 3 and 4). Total wheat germ extracts (lanes 1 and 3) were subjected to affinity chromatography on glutathione-Sepharose beads (lanes 2 and 4). The proteins were separated on denaturing gels and subjected to silver staining or transferred onto a nitrocellulose sheet and reacted with the anti-GST antibody. Molecular masses (kDa) are shown on the left. B, Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer containing [γ-32P]ATP, separated on a denaturing gel, and Coomassie stained (left panel). Autoradiograph of the gel (right panel). Arrows indicate the migration of GST-TgPKA-C and GST-GFP. C, Purified GST-TgPKA-C was incubated in kinase buffer (lane 1). The labeled protein was treated with λ-protein phosphatase (lane 2). Reaction mixtures were then subjected to resolution on an 8% SDS-PAGE gel, followed by Coomassie staining (left panel). Autoradiograph of the gel (C). D, Purified GST-GFP (lane 1) or GST-TgPKA-C (lane 2) was incubated in kinase buffer with Histone IIAS and separated on a 15% denaturing gel followed by Coomassie staining (left panel). Autoradiograph of the gel (right panel). Molecular masses (kDa) are shown on the left.
Mentions: To characterize the function of the protein, we expressed and purified TgPKA-C as a GST fusion protein using a wheat germ cell-free protein synthesis system. Purified protein was then electrophoretically separated in a denaturing gel and either silver-stained or immunoblotted with rabbit antiserum containing α-GST antibody (Figure 3A). The purified proteins from the wheat germ extracts after cell-free protein synthesis with either pEU-GST-GFP [for the expression of GST and green fluorescence protein (GFP) fusion protein] or pEU-GST-TgPKA-C contained one major purified protein with an Mr of 53,000 or 69,000, respectively, as detected by silver staining. These proteins reacted with antiserum containing α-GST antibody. These data indicate that we successfully purified the desired GST fusion proteins.

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