Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein
Pykl (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pykl synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate...
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2006
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02646021_v396_n1_p117_Portela http://hdl.handle.net/20.500.12110/paper_02646021_v396_n1_p117_Portela |
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paper:paper_02646021_v396_n1_p117_Portela2023-06-08T15:23:12Z Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein Kinetics Phosphorylation Protein kinase A (PKA) Pyruvate kinase 1 (Pyk1) Substrate specificity Yeast Fructose Proteins Substrates Yeast Kinetics Phosphorylation Protein kinase A (PKA) Pyruvate kinase 1 (Pyk1) Substrate specificity Enzyme kinetics cyclic AMP dependent protein kinase kemptide mutant protein pyruvate kinase synthetic peptide amino acid sequence article cow gene mutation heart in vitro study in vivo study nonhuman polyacrylamide gel electrophoresis priority journal protein analysis protein phosphorylation protein targeting Saccharomyces cerevisiae yeast Amino Acid Motifs Amino Acid Sequence Animals Cattle Consensus Sequence Cyclic AMP-Dependent Protein Kinases Electrophoresis, Polyacrylamide Gel Fructosediphosphates Kinetics Molecular Sequence Data Mutagenesis, Site-Directed Peptide Fragments Phosphorylation Phosphoserine Protein Binding Protein Processing, Post-Translational Reproducibility of Results Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Substrate Specificity Bovinae Mammalia Saccharomyces cerevisiae Pykl (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pykl synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate for PKA in vitro, with a Ksp* (specificity constant) 10-fold and 3-fold higher than Kemptide for bovine heart and yeast PKA respectively. In vitro phosphorylation of the Pykl S22A mutant protein was decreased by as much as 90% when compared with wild-type Pyk1 and the Pyk1 T94A mutant. The Ksp* values for Pyk1 and Pyk1 T94A were the same, indicating that both proteins are phosphorylated at the same site by PKA. Two-dimensional PAGE of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation prevented the formation of one of the Pyk1 isoforms. We conclude that in yeast the major PKA phosphorylation site of Pyk1 is Ser22. Phosphorylation of Ser22 leads to a Pykl enzyme that is more active in the absence of FBP (fructose 1,6-bisphosphate). The specificity of yeast and mammalian PKA towards the S22 peptide and towards whole Pyk1 protein was measured and compared. The Ksp* for the S22 peptide is higher than that for Pyk1, indicating that the peptide modelled on Pyk1 is a much better substrate than Pyk1, regardless of which tissue was used as the source of PKA. However, the Km of Pyk1 protein is lower than that of the better substrate, the S22 peptide, indicating that ground-state substrate binding is not the major determinant of substrate specificity for PKA. © 2006 Biochemical Society. 2006 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02646021_v396_n1_p117_Portela http://hdl.handle.net/20.500.12110/paper_02646021_v396_n1_p117_Portela |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Kinetics Phosphorylation Protein kinase A (PKA) Pyruvate kinase 1 (Pyk1) Substrate specificity Yeast Fructose Proteins Substrates Yeast Kinetics Phosphorylation Protein kinase A (PKA) Pyruvate kinase 1 (Pyk1) Substrate specificity Enzyme kinetics cyclic AMP dependent protein kinase kemptide mutant protein pyruvate kinase synthetic peptide amino acid sequence article cow gene mutation heart in vitro study in vivo study nonhuman polyacrylamide gel electrophoresis priority journal protein analysis protein phosphorylation protein targeting Saccharomyces cerevisiae yeast Amino Acid Motifs Amino Acid Sequence Animals Cattle Consensus Sequence Cyclic AMP-Dependent Protein Kinases Electrophoresis, Polyacrylamide Gel Fructosediphosphates Kinetics Molecular Sequence Data Mutagenesis, Site-Directed Peptide Fragments Phosphorylation Phosphoserine Protein Binding Protein Processing, Post-Translational Reproducibility of Results Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Substrate Specificity Bovinae Mammalia Saccharomyces cerevisiae |
| spellingShingle |
Kinetics Phosphorylation Protein kinase A (PKA) Pyruvate kinase 1 (Pyk1) Substrate specificity Yeast Fructose Proteins Substrates Yeast Kinetics Phosphorylation Protein kinase A (PKA) Pyruvate kinase 1 (Pyk1) Substrate specificity Enzyme kinetics cyclic AMP dependent protein kinase kemptide mutant protein pyruvate kinase synthetic peptide amino acid sequence article cow gene mutation heart in vitro study in vivo study nonhuman polyacrylamide gel electrophoresis priority journal protein analysis protein phosphorylation protein targeting Saccharomyces cerevisiae yeast Amino Acid Motifs Amino Acid Sequence Animals Cattle Consensus Sequence Cyclic AMP-Dependent Protein Kinases Electrophoresis, Polyacrylamide Gel Fructosediphosphates Kinetics Molecular Sequence Data Mutagenesis, Site-Directed Peptide Fragments Phosphorylation Phosphoserine Protein Binding Protein Processing, Post-Translational Reproducibility of Results Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Substrate Specificity Bovinae Mammalia Saccharomyces cerevisiae Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein |
| topic_facet |
Kinetics Phosphorylation Protein kinase A (PKA) Pyruvate kinase 1 (Pyk1) Substrate specificity Yeast Fructose Proteins Substrates Yeast Kinetics Phosphorylation Protein kinase A (PKA) Pyruvate kinase 1 (Pyk1) Substrate specificity Enzyme kinetics cyclic AMP dependent protein kinase kemptide mutant protein pyruvate kinase synthetic peptide amino acid sequence article cow gene mutation heart in vitro study in vivo study nonhuman polyacrylamide gel electrophoresis priority journal protein analysis protein phosphorylation protein targeting Saccharomyces cerevisiae yeast Amino Acid Motifs Amino Acid Sequence Animals Cattle Consensus Sequence Cyclic AMP-Dependent Protein Kinases Electrophoresis, Polyacrylamide Gel Fructosediphosphates Kinetics Molecular Sequence Data Mutagenesis, Site-Directed Peptide Fragments Phosphorylation Phosphoserine Protein Binding Protein Processing, Post-Translational Reproducibility of Results Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Substrate Specificity Bovinae Mammalia Saccharomyces cerevisiae |
| description |
Pykl (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pykl synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate for PKA in vitro, with a Ksp* (specificity constant) 10-fold and 3-fold higher than Kemptide for bovine heart and yeast PKA respectively. In vitro phosphorylation of the Pykl S22A mutant protein was decreased by as much as 90% when compared with wild-type Pyk1 and the Pyk1 T94A mutant. The Ksp* values for Pyk1 and Pyk1 T94A were the same, indicating that both proteins are phosphorylated at the same site by PKA. Two-dimensional PAGE of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation prevented the formation of one of the Pyk1 isoforms. We conclude that in yeast the major PKA phosphorylation site of Pyk1 is Ser22. Phosphorylation of Ser22 leads to a Pykl enzyme that is more active in the absence of FBP (fructose 1,6-bisphosphate). The specificity of yeast and mammalian PKA towards the S22 peptide and towards whole Pyk1 protein was measured and compared. The Ksp* for the S22 peptide is higher than that for Pyk1, indicating that the peptide modelled on Pyk1 is a much better substrate than Pyk1, regardless of which tissue was used as the source of PKA. However, the Km of Pyk1 protein is lower than that of the better substrate, the S22 peptide, indicating that ground-state substrate binding is not the major determinant of substrate specificity for PKA. © 2006 Biochemical Society. |
| title |
Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein |
| title_short |
Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein |
| title_full |
Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein |
| title_fullStr |
Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein |
| title_full_unstemmed |
Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein |
| title_sort |
characterization of yeast pyruvate kinase 1 as a protein kinase a substrate, and specificity of the phosphorylation site sequence in the whole protein |
| publishDate |
2006 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02646021_v396_n1_p117_Portela http://hdl.handle.net/20.500.12110/paper_02646021_v396_n1_p117_Portela |
| _version_ |
1768545971618185216 |