Characterization of pyruvate kinae 1 of Saccharomyces cerevisiae as a PKA substrate

PORTELA, P., MORENO, S. AND ROSSI, S.

Iguazu, Misiones

Congreso; XL Reunion Anual de SAIB; 2004

SAIB

In this work we characterized a S.cerevsiae PKA substrate, through the identification of the phosphorylation site, and the study of how this phosphorylation affects its activity and how a natural substrate participates in yeast holoenzyme activation. Analysis of the Pykl sequence revealed three potential phosphorylation sites (Ser22, Thr94 and Thr478) within a PKA sequence motif. Three peptides containing these sites were synthesized .In vitro assays using catalytic subunit from bovine heart PKA (Cb) indicate that only one peptide, the LRRTSIIGTR, incIuding the Ser22, was substrate for the kinase. The specificity constant (kc./Km) is 6.6 pmol (min.unittlJ.1M'1, one order lower than the one for kemptide. The peptides behavior was the same when assayed in vitro with crude extracts from a TPKI tpk2L1tpk3L1bcyl L1 strain as kinase source. In vivo, phosphorylation using permeabilized yeast ceIls, indicates too that the only peptide Ser22 was substrate. Pykl S22A and PyklT94A mutant proteins were expressed in a yeast strain whith a mutation that prevents endogenous Pykl expression (PYKl-5) and were subjected to phosphorylation with Cb and TPKI. 0.5 and 0.075 mol of phosphate were incorporated in Pykl respectively and it was reduced by more than 90% in PykIS22A. The kc./Km ofCb for Pykl, and Pyk}T94A were in the same order. The phosphorylation in the Ser22 is important in the regulation ofthe activity ófPykl.Preliminary results indicates a participation of the ,substrate in the mechanism of activation of yeast PKA holoenzyme. Peptide Ser22 produced a decreased in the AO.5 and is a better activator as compared withpyk whole protein which is similar to kemptide.