INVESTIGADORES
PORTELA Paula
artículos
Título:
Characterization of substrates that have differential effect on Saccharomyces cerevisiae protein kinase A
Autor/es:
GALELLO, F., PORTELA, P., MORENO, S. AND ROSSI, S.
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Referencias:
Año: 2010 p. 1 - 5
ISSN:
0021-9258
Resumen:
The specificity in the 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 sec-1. We analyze the substrate behavior and
sequence determinants around the phosphorylation
site of three protein substrates, Pyruvate kinase 1
(Pyk1), Pyruvate kinase 2 (Pyk2) and Neutral
Trehalase (Nth1). Nth1 protein is a better substrate
than Pyk1 protein and both are phosphorylated by
either Tpk1 or Tpk2. Both enzymes have also 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.