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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Autores principales: Galello, F., Portela, P., Moreno, S., Rossi, S.
Formato: Artículo publishedVersion
Publicado: 2010
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Acceso en línea:http://hdl.handle.net/20.500.12110/paper_00219258_v285_n39_p29770_Galello
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_00219258_v285_n39_p29770_Galello_oai
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id I28-R145-paper_00219258_v285_n39_p29770_Galello_oai
record_format dspace
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (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, F.
Portela, P.
Moreno, S.
Rossi, S.
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.
format Artículo
Artículo
publishedVersion
author Galello, F.
Portela, P.
Moreno, S.
Rossi, S.
author_facet Galello, F.
Portela, P.
Moreno, S.
Rossi, S.
author_sort Galello, F.
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 http://hdl.handle.net/20.500.12110/paper_00219258_v285_n39_p29770_Galello
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_00219258_v285_n39_p29770_Galello_oai
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AT rossis characterizationofsubstratesthathaveadifferentialeffectonsaccharomycescerevisiaeproteinkinaseaholoenzymeactivation
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spelling I28-R145-paper_00219258_v285_n39_p29770_Galello_oai2020-10-19 Galello, F. Portela, P. Moreno, S. Rossi, S. 2010 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. application/pdf http://hdl.handle.net/20.500.12110/paper_00219258_v285_n39_p29770_Galello info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar J. Biol. Chem. 2010;285(39):29770-29779 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 Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation info:eu-repo/semantics/article info:ar-repo/semantics/artículo info:eu-repo/semantics/publishedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_00219258_v285_n39_p29770_Galello_oai