Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation
The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits i...
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2010
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219258_v285_n39_p29770_Galello http://hdl.handle.net/20.500.12110/paper_00219258_v285_n39_p29770_Galello |
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paper:paper_00219258_v285_n39_p29770_Galello2023-06-08T14:43:31Z Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation Galello, Fiorella Ariadna Portela, Paula Moreno de Colonna, Silvia Rossi, Silvia Graciela Catalysis Catalysts Enzymes Fetal monitoring Mammals Molecular biology Molecular recognition Peptides Phosphorylation Yeast Acidic residues Catalytic reactions Catalytic subunits Catalytic turnover Coactivators Consensus motif Differential effect Hydrophobic residues Isoforms N-terminals Peptide arrays Peptide substrates Phosphorylation sites Protein kinase A Protein substrate Regulatory subunits Saccharomyces cerevisiae Sequence determinants Substrate effects Target sequences Substrates arginine cyclic AMP dependent protein kinase holoenzyme neutral trehalase 1 protein Bcy1 protein subunit protein Tpk1 protein Tpk2 pyruvate kinase 1 pyruvate kinase 2 serine unclassified drug cyclic AMP dependent protein kinase focal adhesion kinase 2 holoenzyme Saccharomyces cerevisiae protein TPK2 protein, S cerevisiae amino acid sequence amino terminal sequence article carboxy terminal sequence consensus sequence controlled study enzyme activation enzyme active site enzyme kinetics hydrophobicity nonhuman priority journal protein motif protein phosphorylation Saccharomyces cerevisiae steady state enzyme specificity enzymology genetics metabolism phosphorylation physiology Saccharomyces cerevisiae Mammalia Saccharomyces cerevisiae Amino Acid Motifs Catalytic Domain Cyclic AMP-Dependent Protein Kinases Enzyme Activation Focal Adhesion Kinase 2 Holoenzymes Phosphorylation Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Substrate Specificity The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits isoforms Tpk1 and Tpk2 were determined, and both enzymes are shown to have the same value of 3 s-1. We analyze the substrate behavior and sequence determinants around the phosphorylation site of three protein substrates, Pyk1, Pyk2, and Nth1. Nth1 protein is a better substrate than Pyk1 protein, and both are phosphorylated by either Tpk1 or Tpk2. Both enzymes also have the same selectivity toward the protein substrates and the peptides derived from them. The three substrates contain one or more Arg-Arg-X-Ser consensus motif, but not all of them are phosphorylated. The determinants for specificity were studied using the peptide arrays. Acidic residues in the position P+1 or in the N-terminal flank are deleterious, and positive residues present beyond P-2 and P-3 favor the catalytic reaction. A bulky hydrophobic residue in position P+1 is not critical. The best substrate has in position P+4 an acidic residue, equivalent to the one in the inhibitory sequence of Bcy1, the yeast regulatory subunit of PKA. The substrate effect in the holoenzyme activation was analyzed, and we demonstrate that peptides and protein substrates sensitized the holoenzyme to activation by cAMP in different degrees, depending on their sequences. The results also suggest that protein substrates are better co-activators than peptide substrates. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Fil:Galello, F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Portela, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Moreno, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Rossi, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2010 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219258_v285_n39_p29770_Galello http://hdl.handle.net/20.500.12110/paper_00219258_v285_n39_p29770_Galello |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Catalysis Catalysts Enzymes Fetal monitoring Mammals Molecular biology Molecular recognition Peptides Phosphorylation Yeast Acidic residues Catalytic reactions Catalytic subunits Catalytic turnover Coactivators Consensus motif Differential effect Hydrophobic residues Isoforms N-terminals Peptide arrays Peptide substrates Phosphorylation sites Protein kinase A Protein substrate Regulatory subunits Saccharomyces cerevisiae Sequence determinants Substrate effects Target sequences Substrates arginine cyclic AMP dependent protein kinase holoenzyme neutral trehalase 1 protein Bcy1 protein subunit protein Tpk1 protein Tpk2 pyruvate kinase 1 pyruvate kinase 2 serine unclassified drug cyclic AMP dependent protein kinase focal adhesion kinase 2 holoenzyme Saccharomyces cerevisiae protein TPK2 protein, S cerevisiae amino acid sequence amino terminal sequence article carboxy terminal sequence consensus sequence controlled study enzyme activation enzyme active site enzyme kinetics hydrophobicity nonhuman priority journal protein motif protein phosphorylation Saccharomyces cerevisiae steady state enzyme specificity enzymology genetics metabolism phosphorylation physiology Saccharomyces cerevisiae Mammalia Saccharomyces cerevisiae Amino Acid Motifs Catalytic Domain Cyclic AMP-Dependent Protein Kinases Enzyme Activation Focal Adhesion Kinase 2 Holoenzymes Phosphorylation Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Substrate Specificity |
| spellingShingle |
Catalysis Catalysts Enzymes Fetal monitoring Mammals Molecular biology Molecular recognition Peptides Phosphorylation Yeast Acidic residues Catalytic reactions Catalytic subunits Catalytic turnover Coactivators Consensus motif Differential effect Hydrophobic residues Isoforms N-terminals Peptide arrays Peptide substrates Phosphorylation sites Protein kinase A Protein substrate Regulatory subunits Saccharomyces cerevisiae Sequence determinants Substrate effects Target sequences Substrates arginine cyclic AMP dependent protein kinase holoenzyme neutral trehalase 1 protein Bcy1 protein subunit protein Tpk1 protein Tpk2 pyruvate kinase 1 pyruvate kinase 2 serine unclassified drug cyclic AMP dependent protein kinase focal adhesion kinase 2 holoenzyme Saccharomyces cerevisiae protein TPK2 protein, S cerevisiae amino acid sequence amino terminal sequence article carboxy terminal sequence consensus sequence controlled study enzyme activation enzyme active site enzyme kinetics hydrophobicity nonhuman priority journal protein motif protein phosphorylation Saccharomyces cerevisiae steady state enzyme specificity enzymology genetics metabolism phosphorylation physiology Saccharomyces cerevisiae Mammalia Saccharomyces cerevisiae Amino Acid Motifs Catalytic Domain Cyclic AMP-Dependent Protein Kinases Enzyme Activation Focal Adhesion Kinase 2 Holoenzymes Phosphorylation Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Substrate Specificity Galello, Fiorella Ariadna Portela, Paula Moreno de Colonna, Silvia Rossi, Silvia Graciela Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation |
| topic_facet |
Catalysis Catalysts Enzymes Fetal monitoring Mammals Molecular biology Molecular recognition Peptides Phosphorylation Yeast Acidic residues Catalytic reactions Catalytic subunits Catalytic turnover Coactivators Consensus motif Differential effect Hydrophobic residues Isoforms N-terminals Peptide arrays Peptide substrates Phosphorylation sites Protein kinase A Protein substrate Regulatory subunits Saccharomyces cerevisiae Sequence determinants Substrate effects Target sequences Substrates arginine cyclic AMP dependent protein kinase holoenzyme neutral trehalase 1 protein Bcy1 protein subunit protein Tpk1 protein Tpk2 pyruvate kinase 1 pyruvate kinase 2 serine unclassified drug cyclic AMP dependent protein kinase focal adhesion kinase 2 holoenzyme Saccharomyces cerevisiae protein TPK2 protein, S cerevisiae amino acid sequence amino terminal sequence article carboxy terminal sequence consensus sequence controlled study enzyme activation enzyme active site enzyme kinetics hydrophobicity nonhuman priority journal protein motif protein phosphorylation Saccharomyces cerevisiae steady state enzyme specificity enzymology genetics metabolism phosphorylation physiology Saccharomyces cerevisiae Mammalia Saccharomyces cerevisiae Amino Acid Motifs Catalytic Domain Cyclic AMP-Dependent Protein Kinases Enzyme Activation Focal Adhesion Kinase 2 Holoenzymes Phosphorylation Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Substrate Specificity |
| description |
The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits isoforms Tpk1 and Tpk2 were determined, and both enzymes are shown to have the same value of 3 s-1. We analyze the substrate behavior and sequence determinants around the phosphorylation site of three protein substrates, Pyk1, Pyk2, and Nth1. Nth1 protein is a better substrate than Pyk1 protein, and both are phosphorylated by either Tpk1 or Tpk2. Both enzymes also have the same selectivity toward the protein substrates and the peptides derived from them. The three substrates contain one or more Arg-Arg-X-Ser consensus motif, but not all of them are phosphorylated. The determinants for specificity were studied using the peptide arrays. Acidic residues in the position P+1 or in the N-terminal flank are deleterious, and positive residues present beyond P-2 and P-3 favor the catalytic reaction. A bulky hydrophobic residue in position P+1 is not critical. The best substrate has in position P+4 an acidic residue, equivalent to the one in the inhibitory sequence of Bcy1, the yeast regulatory subunit of PKA. The substrate effect in the holoenzyme activation was analyzed, and we demonstrate that peptides and protein substrates sensitized the holoenzyme to activation by cAMP in different degrees, depending on their sequences. The results also suggest that protein substrates are better co-activators than peptide substrates. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. |
| author |
Galello, Fiorella Ariadna Portela, Paula Moreno de Colonna, Silvia Rossi, Silvia Graciela |
| author_facet |
Galello, Fiorella Ariadna Portela, Paula Moreno de Colonna, Silvia Rossi, Silvia Graciela |
| author_sort |
Galello, Fiorella Ariadna |
| title |
Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation |
| title_short |
Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation |
| title_full |
Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation |
| title_fullStr |
Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation |
| title_full_unstemmed |
Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation |
| title_sort |
characterization of substrates that have a differential effect on saccharomyces cerevisiae protein kinase a holoenzyme activation |
| publishDate |
2010 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219258_v285_n39_p29770_Galello http://hdl.handle.net/20.500.12110/paper_00219258_v285_n39_p29770_Galello |
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| _version_ |
1768546524016410624 |