Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro.

Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and PKA-2). The presence of PKA in both fractions was confirmed by immunoblotting with anti-Bcy1 antibodies. Yeast pyruvate kinase Pyk1 identified by amino acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodies copurified with the PKA-1 but not the -2 fraction. Pyk1 can be phosphorylated by yeast PKA in vitro in the presence of cAMP and cGMP. Two-dimensional gel electrophoretic analysis revealed four phosphorylated forms of Pyk1 modified by PKA. In phosphorylation of Pyk1 mainly the Tpk2 catalytic subunit of yeast PKA was involved.

Phosphorylation of yeast ribosomal proteins by CKI and CKII in the presence of heparin.

We have found that heparin has a different effect on Trichosporon cutaneum ribosomal protein phosphorylation by CKI and by CKII. In the presence of heparin, modification of 13 kDa, 19 kDa and 38 kDa proteins catalyzed by CKII was inhibited, while in the case of CKI, in addition to protein of 15 kDa, phosphorylation of 20 kDa and 35 kDa proteins was detected. It was also found that, in the presence of heparin, phosphorylation of P proteins (13 kDa and 38 kDa) by ribosome-bound protein kinases was inhibited. Moreover at the same conditions modification of 40 kDa protein was observed in all four yeast species tested.

PKA from Saccharomyces cerevisiae can be activated by cyclic AMP and cyclic GMP.

Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms. Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified from S. cerevisiae was almost equally activated by cAMP and cGMP in 3 x 10(-6) M concentrations of either nucleotide in the presence of Mg2+ ions. Interestingly, if Mn2+ ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase. Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, while N6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent. It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion. The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.

The effect of heparin on the activity of Trichosporon cutaneum casein kinase I.

Casein kinase I from Trichosporon cutaneum ribosome-free extracts was purified. Its molecular mass was calculated for 33 kDa. It was shown that casein, phosvitin and Trichosporon cutaneum ribosomal protein of 15 kDa were preferable substrates for the enzyme. It was found that heparin can stimulate or inhibit CKI activity depending on the substrate used. Stimulation of casein and inhibition of phosvitin phosphorylation was observed. In addition it was shown that ribosomal proteins of 19 kDa and 38 kDa were phosphorylated by CKI only in the presence of heparin.