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 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.
Galello
Fiorella
F
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428 Buenos Aires, Argentina.
Portela
Paula
P
Moreno
Silvia
S
Rossi
Silvia
S
D020816:Amino Acid Motifs; D020134:Catalytic Domain / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D051418:Focal Adhesion Kinase 2 / Q000235:genetics / Q000378:metabolism; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology
D020035:Holoenzymes; D029701:Saccharomyces cerevisiae Proteins; D051418:Focal Adhesion Kinase 2; D017868:Cyclic AMP-Dependent Protein Kinases; C457179:TPK2 protein, S cerevisiae