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Abstract:

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.

Registro:

Documento: Artículo
Título:Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein
Autor:Portela, P.; Moreno, S.; Rossi, S.
Filiación:Departamento de Química Biológica, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428 Buenos Aires, Argentina
Palabras clave: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
Año:2006
Volumen:396
Número:1
Página de inicio:117
Página de fin:126
DOI: http://dx.doi.org/10.1042/BJ20051642
Título revista:Biochemical Journal
Título revista abreviado:Biochem. J.
ISSN:02646021
CODEN:BIJOA
CAS:kemptide, 65189-71-1; pyruvate kinase, 9001-59-6; Cyclic AMP-Dependent Protein Kinases, EC 2.7.1.37; fructose-1,6-diphosphate, 488-69-7; Fructosediphosphates; Peptide Fragments; Phosphoserine, 17885-08-4; Saccharomyces cerevisiae Proteins
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02646021_v396_n1_p117_Portela

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Citas:

---------- APA ----------
Portela, P., Moreno, S. & Rossi, S. (2006) . Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein. Biochemical Journal, 396(1), 117-126.
http://dx.doi.org/10.1042/BJ20051642
---------- CHICAGO ----------
Portela, P., Moreno, S., Rossi, S. "Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein" . Biochemical Journal 396, no. 1 (2006) : 117-126.
http://dx.doi.org/10.1042/BJ20051642
---------- MLA ----------
Portela, P., Moreno, S., Rossi, S. "Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein" . Biochemical Journal, vol. 396, no. 1, 2006, pp. 117-126.
http://dx.doi.org/10.1042/BJ20051642
---------- VANCOUVER ----------
Portela, P., Moreno, S., Rossi, S. Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein. Biochem. J. 2006;396(1):117-126.
http://dx.doi.org/10.1042/BJ20051642