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Papers In Press, published online ahead of print June 12, 2002
Departamento de Quimica Biologica, Universidad de Buenos Aires, Buenos Aires 1428
Corresponding Author: srossi{at}qb.fcen.uba.ar
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 GST-Pyk1 and GST-Pyk2 were phosphorylated in vitro by bovine heart 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 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-biphosphate 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, while in the presence of fructose-1,6-biphosphate it shows an nH of 1.4, as compared to an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.
J. Biol. Chem, 10.1074/jbc.M201094200
Submitted on February 1, 2002
Revised on June 4, 2002
Accepted on June 12, 2002
In vivo and in vitro phosphorylation of two isoforms of yeast pyruvate kinase by protein kinase A
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