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J. Biol. Chem., Vol. 277, Issue 34, 30477-30487, August 23, 2002
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From the § Laboratory of Protein Structure,
National Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, United Kingdom and the Saccharomyces cerevisiae
pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an
immunoprecipitate of yeast protein kinase A holoenzyme
(HA-Tpk1·Bcy1) and to be phosphorylated in a
cAMP-dependent process. Both glutathione
S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated
in vitro by the bovine heart protein kinase A (PKA)
catalytic subunit and by immobilized yeast HA-Tpk1. The specificity
constant for the phosphorylation of GST-Pyk1 and GST-Pyk2 by bovine
catalytic subunit was in the range of the value for
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were
phosphorylated in vivo, in intact cells overexpressing the
protein, or in vitro using crude extracts, as source of
protein kinase A, when a wild type strain was used but were not
phosphorylated when using a strain with only one TPK gene
with an attenuated mutation (tpk1w1). The effect of
phosphorylation on Pyk activity was assayed in partially purified
preparations from three strains, containing different endogenous
protein kinase A activity levels. Pyk1 activity was measured at
different phosphoenolpyruvate concentrations in the absence or in the
presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus
those from the lowest protein kinase A activity indicate that the
enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for
phosphoenolpyruvate to the left and an increase in the Hill
coefficient, whereas in the presence of fructose 1,6-bisphosphate it
shows an nH value of 1.4, as compared with an
nH of 2 for the Pyk1 obtained from extracts
with almost null protein kinase A activity.
In Vivo and in Vitro Phosphorylation of
Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A*
,, and¶
Departamento de
Química Biológica, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires, Ciudad Universitaria,
Pabellón 2, Buenos Aires 1428, Argentina
*
This research was supported in part by grants from the
University of Buenos Aires and Ministerio de Salud through the
Carrillo-Oñativia Fellowship.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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