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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
Identification of TgPKA-C.Comparison of predicted TgPKA-C amino acid sequences (ToxoDB identifier; TGGT1_081170) with those of P. falciparum PKA-C (PfPKA-C, GenBank Accession Number: AAB70118) and H. sapiens PKA-Cα (HsPKA-Cα, GenBank Accession Number: NP_002721). Amino acid identity (black boxes) and similarity (gray boxes) are shown within the protein kinase domain. Gaps introduced to optimize alignments are marked by dashes. Roman numerals indicate the 11 conserved protein kinase subdomains [23]. The 12 most highly conserved residues are highlighted with filled triangles. The sequence thought to be necessary for binding to mammalian PKI is shown with a dotted line.
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pone-0022492-g001: Identification of TgPKA-C.Comparison of predicted TgPKA-C amino acid sequences (ToxoDB identifier; TGGT1_081170) with those of P. falciparum PKA-C (PfPKA-C, GenBank Accession Number: AAB70118) and H. sapiens PKA-Cα (HsPKA-Cα, GenBank Accession Number: NP_002721). Amino acid identity (black boxes) and similarity (gray boxes) are shown within the protein kinase domain. Gaps introduced to optimize alignments are marked by dashes. Roman numerals indicate the 11 conserved protein kinase subdomains [23]. The 12 most highly conserved residues are highlighted with filled triangles. The sequence thought to be necessary for binding to mammalian PKI is shown with a dotted line.

Mentions: The amino acid sequence alignment of the putative TgPKA-C (ToxoDB identifier; TGGT1_081170), which we identified for the first time, is shown in Figure 1 together with those of P. falciparum and Homo sapiens. The putative TgPKA-C (343 amino acid residues) showed 54% identity with H. sapiens PKA-Cα (HsPKA-Cα, GenBank Accession Number: NP_002721) and 57% identity with P. falciparum PKA-C (PfPKA-C, GenBank Accession Number: AAB70118). A multiple alignment using the ClustalW program showed that the 11 major subdomains of protein kinases (I–XI) [23] are conserved in the amino acid sequence of TgPKA-C. Moreover, highly conserved individual amino acids that are involved in ATP binding, peptide binding, stabilizing, or autophosphorylation were also seen in TgPKA-C amino acid sequences [24], [25].


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)

Identification of TgPKA-C.Comparison of predicted TgPKA-C amino acid sequences (ToxoDB identifier; TGGT1_081170) with those of P. falciparum PKA-C (PfPKA-C, GenBank Accession Number: AAB70118) and H. sapiens PKA-Cα (HsPKA-Cα, GenBank Accession Number: NP_002721). Amino acid identity (black boxes) and similarity (gray boxes) are shown within the protein kinase domain. Gaps introduced to optimize alignments are marked by dashes. Roman numerals indicate the 11 conserved protein kinase subdomains [23]. The 12 most highly conserved residues are highlighted with filled triangles. The sequence thought to be necessary for binding to mammalian PKI is shown with a dotted line.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022492-g001: Identification of TgPKA-C.Comparison of predicted TgPKA-C amino acid sequences (ToxoDB identifier; TGGT1_081170) with those of P. falciparum PKA-C (PfPKA-C, GenBank Accession Number: AAB70118) and H. sapiens PKA-Cα (HsPKA-Cα, GenBank Accession Number: NP_002721). Amino acid identity (black boxes) and similarity (gray boxes) are shown within the protein kinase domain. Gaps introduced to optimize alignments are marked by dashes. Roman numerals indicate the 11 conserved protein kinase subdomains [23]. The 12 most highly conserved residues are highlighted with filled triangles. The sequence thought to be necessary for binding to mammalian PKI is shown with a dotted line.
Mentions: The amino acid sequence alignment of the putative TgPKA-C (ToxoDB identifier; TGGT1_081170), which we identified for the first time, is shown in Figure 1 together with those of P. falciparum and Homo sapiens. The putative TgPKA-C (343 amino acid residues) showed 54% identity with H. sapiens PKA-Cα (HsPKA-Cα, GenBank Accession Number: NP_002721) and 57% identity with P. falciparum PKA-C (PfPKA-C, GenBank Accession Number: AAB70118). A multiple alignment using the ClustalW program showed that the 11 major subdomains of protein kinases (I–XI) [23] are conserved in the amino acid sequence of TgPKA-C. Moreover, highly conserved individual amino acids that are involved in ATP binding, peptide binding, stabilizing, or autophosphorylation were also seen in TgPKA-C amino acid sequences [24], [25].

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