IADR Abstract Archives
Active Motifs and Function of Ptk1 in Porphyromonas gingivalis
Objectives: Porphyromonas gingivalis engages in polymicrobial synergy with Streptococcus gordonii. Attachment of P. gingivalis to S. gordonii, and extracellular polysaccharide (EPS) production, are controlled by a P. gingivalis protein tyrosine kinase 1 (Ptk1). However, the nature of the signaling cascade regulated by Ptk1 is poorly understood. In this study we identified active motifs and substrates of Ptk1 in P. gingivalis.
Methods: Site-specific mutations were introduced into functional protein tyrosine kinase 1 (Fptk1). Fptk1 and its derivatives were tested for autophosphorylation, kinase activity for the substrate PGN_0224, a UDP-acetyl-mannosamine dehydrogenase, and transfer of phosphate to transcriptional regulators annotated in the P. gingivalis genome. FLAG labeled Ptk1 was constructed and the subcellular location of the protein determined by Western blotting, ELISA and immunofluorescence.
Results: We found that ExxRxxR[S/T], Walker A, and Walker B motifs were catalytic domains of Fptk1 for both autophosphorylation and substrate phosphorylation. The C-terminal Y-cluster was the main autophosphorylation site in Ptk1. We also demonstrated that Ptk1 is located in the cell membrane, cytoplasm and extracellularly. Ptk1 was able to transfer phosphate groups to the tyrosine residues of transcriptional regulators PGN_0775 and PGN_1019 in P. gingivalis.
Conclusions: We conclude that the ExxRxxR[S/T], Walker A, Walker B and C-terminal Y-cluster are essential functional domains for catalytic activity of Ptk1. The Ptk1 protein is both secreted and widely distributed within the bacterial cells. The versatility of Ptk1, which lacks eukaryotic homologs, may provide the basis for the development of novel drug targets targeted against P. gingivalis.
Methods: Site-specific mutations were introduced into functional protein tyrosine kinase 1 (Fptk1). Fptk1 and its derivatives were tested for autophosphorylation, kinase activity for the substrate PGN_0224, a UDP-acetyl-mannosamine dehydrogenase, and transfer of phosphate to transcriptional regulators annotated in the P. gingivalis genome. FLAG labeled Ptk1 was constructed and the subcellular location of the protein determined by Western blotting, ELISA and immunofluorescence.
Results: We found that ExxRxxR[S/T], Walker A, and Walker B motifs were catalytic domains of Fptk1 for both autophosphorylation and substrate phosphorylation. The C-terminal Y-cluster was the main autophosphorylation site in Ptk1. We also demonstrated that Ptk1 is located in the cell membrane, cytoplasm and extracellularly. Ptk1 was able to transfer phosphate groups to the tyrosine residues of transcriptional regulators PGN_0775 and PGN_1019 in P. gingivalis.
Conclusions: We conclude that the ExxRxxR[S/T], Walker A, Walker B and C-terminal Y-cluster are essential functional domains for catalytic activity of Ptk1. The Ptk1 protein is both secreted and widely distributed within the bacterial cells. The versatility of Ptk1, which lacks eukaryotic homologs, may provide the basis for the development of novel drug targets targeted against P. gingivalis.