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.

Phosphorylation of ribosomal proteins by ribosome-associated protein kinases of Trichosporon cutaneum.

Four ribosomal proteins of Mr 13 kDa, 15 kDa, 19 kDa and 38 kDa were identified as phosphorylation substrates for protein kinases tightly associated with Trichosporon cutaneum ribosomes. It was found that proteins of 13 kDa, 19 kDa and 38 kDa were phosphorylated by multifunctional casein kinase II while the protein of 15 kDa by casein kinase I. Proteins of 13 kDa and 38 kDa were detected in the large subunits while 15 kDa and 19 kDa in the small ribosomal subunits. By using isoelectrofocusing the protein of 13 kDa was resolved into two individual phosphorylated forms. The phosphorylation level of both forms was much higher in ribosomes from the cells collected at the exponential growth phase than in those from the stationary phase. The same phosphoprotein forms were identified in ribosomes from in vitro and in vivo [32P]-labelling experiments.

The acidic ribosomal proteins of different yeast species. Phosphorylation by ribosome-associated protein kinases.

Two major ribosomal proteins of Mr 13 kDa and 38 kDa were identified as phosphorylation substrates for ribosome-bound protein kinases from different yeast species. The phosphorylation level was much higher in ribosomes from the cells collected at the exponential growth phase, than in those from the stationary phase. Isoelectrofocusing of the protein band of 13 kDa and subsequent silver staining allowed to identify from four in the case of Pichia stipitis up to ten in Saccharomyces cerevisiae. Saccharomycodes Ludwigii, Torulopsis utilis and Kloeckera apiculata individual peptides. In the most of the yeast species studies five phosphorylated peptides were observed. However, only one or two such phosphopeptides were detected in Pichia stipitis and Trichosporon cutaneum ribosomes, respectively. The same phosphoprotein forms were identified in the in vivo 32P-labelling experiments.

Phosphorylation of acidic ribosomal proteins by ribosome-associated protein kinases of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Two proteins of 13 kDa and 38 kDa, the components of 60S ribosomal subunits, were identified as phosphorylation substrates for protein kinases tightly associated with S. cerevisiae and Schizosaccharomyces pombe ribosomes. An enzyme with properties of multifunctional casein kinase II was detected in ribosome preparations from both yeast species. In S. cerevisiae another protein kinase with high substrate specificity toward those proteins was also identified. By using isoelectric focusing, the protein band of 13 kDa from S. cerevisiae and S. pombe was resolved respectively into three and four major forms of different charge. The same protein forms were phosphorylated in the in vivo 32P-labelling experiments.