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Originally published In Press as doi:10.1074/jbc.M201094200 on June 12, 2002

J. Biol. Chem., Vol. 277, Issue 34, 30477-30487, August 23, 2002
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In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A*

Paula PortelaDagger , Steven Howell§, Silvia MorenoDagger , and Silvia RossiDagger

From the § Laboratory of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom and the Dagger  Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires 1428, Argentina

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.

* 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.

To whom correspondence should be addressed. Tel./Fax: 54-11-4576-3342; E-mail: srossi@qb.fcen.uba.ar.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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