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Abstract:

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.

Registro:

Documento: Artículo
Título:Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation
Autor:Galello, F.; Portela, P.; Moreno, S.; Rossi, S.
Filiación:Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, 1428 Buenos Aires, Argentina
Palabras clave: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
Año:2010
Volumen:285
Número:39
Página de inicio:29770
Página de fin:29779
DOI: http://dx.doi.org/10.1074/jbc.M110.120378
Título revista:Journal of Biological Chemistry
Título revista abreviado:J. Biol. Chem.
ISSN:00219258
CODEN:JBCHA
CAS:arginine, 1119-34-2, 15595-35-4, 7004-12-8, 74-79-3; cyclic AMP dependent protein kinase; serine, 56-45-1, 6898-95-9; Cyclic AMP-Dependent Protein Kinases; Focal Adhesion Kinase 2; Holoenzymes; Saccharomyces cerevisiae Proteins; TPK2 protein, S cerevisiae
PDF:https://bibliotecadigital.exactas.uba.ar/download/paper/paper_00219258_v285_n39_p29770_Galello.pdf
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219258_v285_n39_p29770_Galello

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Citas:

---------- APA ----------
Galello, F., Portela, P., Moreno, S. & Rossi, S. (2010) . Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation. Journal of Biological Chemistry, 285(39), 29770-29779.
http://dx.doi.org/10.1074/jbc.M110.120378
---------- CHICAGO ----------
Galello, F., Portela, P., Moreno, S., Rossi, S. "Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation" . Journal of Biological Chemistry 285, no. 39 (2010) : 29770-29779.
http://dx.doi.org/10.1074/jbc.M110.120378
---------- MLA ----------
Galello, F., Portela, P., Moreno, S., Rossi, S. "Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation" . Journal of Biological Chemistry, vol. 285, no. 39, 2010, pp. 29770-29779.
http://dx.doi.org/10.1074/jbc.M110.120378
---------- VANCOUVER ----------
Galello, F., Portela, P., Moreno, S., Rossi, S. Characterization of substrates that have a differential effect on Saccharomyces cerevisiae protein kinase A holoenzyme activation. J. Biol. Chem. 2010;285(39):29770-29779.
http://dx.doi.org/10.1074/jbc.M110.120378