Identification of PKA phosphorylation site in pyruvate kinase 1 of Saccharomyces cerevisiae

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

Rio de Janeiro, Brasil

Congreso; 11th International Congress on Yeasts; 2004

Yeast pyruvate kinase 1 (Pyk 1) catalyses the irreversible conversion of phosphoenolpyruvate (PEP) to pyruvate, the final step in glycolysis. We have recently demonstrated it is a substrate of yeast protein kinase A (PKA), both in vitro and in vivo. Indirect evidence suggested phosphorylation by PKA increased Pyk activity. In this work we identify the target site of PKA phosphorylation. Analysis of the Pyk I sequence revealed three potential phosphorylation sites (Ser22, Thr94 and Thr478) within a PKA sequence motif. Three peptides containing the potential target sites were synthesized based on the deduced protein sequence. The ability of the peptides as PKA substrates was compared with Kemptide. [n vitro assays using catalytic subunit from bovine heart PI<.A (Cl,) indicate that only one peptide, the LRRTSTlGTR, which contains the Ser22, was substrate for the kinase. The specificity constant (k<a/KIII) expressed as pmol.(ll1in.unit)"' M'I is 6.6, one order lower than the one for kemptide. The peptides' behavior was the sall1e when assayed in vitro with crude extracts from a TPI<.llpk] Ipk3 be)'1 strain as kinase source. [n vivo. phosphorylation using permeabilized yeast cells. indicates that, as in in vitro assays, the only peptide phosphorylated was the one containing Ser22. These results led to the construction of Pyk 1 S22A and Pyk I T~4A ll1utant proteins, expressed in a yeast strain which harbors a mutation that prevents endogenous Pyk 1 expression (P YK 1-5). Pyk 1, Pyk ¡ mA and Pyk I T94A purified proteins were subjected to phosphorylation with Ch. At the point of ll1axill1ul11 phosphorylation, C!J catalyzed the incorporation of 0.3 11101 of phosphate/ll1ol of Pyk in Pyk 1 and Pyk I T94A and only 0.03 mol of phosphate Imol of Pyk in PykIS22A. The kco/KIII of Ch for Pykl, and PyklT94A were 0.068, 0.071 pmol.(min.unitrl M-1 respectively. The kinetic parameters of Pyk l phospborylation were tbe same when using TPK 1 as source of PKA catalytic subunit. These results indicate that Ser 22 is the only target site for PKA. Pyk activity was measured in extracts frol11 P YK 1-5 overexpressing Pyk 1 or Pyk 1 S22A and kinetic parameters determined. The results confirm that the absence of PI<.A phospborylation in Pyk 1 S22A decreased the Pyk activity.