Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro

Cytrynska, M

doi:10.1016/s0378-1097(01)00354-8

Cited by 8 publications

(12 citation statements)

References 16 publications

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“…Preliminary results from Cytryñ ska et al (8) and Rayner et al (9) showed that Pyk1 could be phosphorylated in vitro by Tpk2 and Tpk1, respectively. From our last results and from the results of the immunoprecipitation (Figs.…”

Section: Resultsmentioning

confidence: 99%

“…The stoichiome- tries of phosphorylation attained with C b in vitro are compatible with one phosphorylation site per molecule of pyruvate kinase; however, optimization of the phosphorylation conditions, so as to compensate for the low efficiency usually displayed by PKAs, was not assayed. It has been reported that Pyk1 could be visualized by isoelectric focusing as four different forms with different isoelectric points, suggesting multiple phosphorylation sites per monomer, either under in vitro phosphorylation conditions (8) or analyzing the protein profile from exponentially growing cells (35). In higher eukaryotes at least, several kinases have been reported to be responsible for the multiphosphorylation of pyruvate kinase (36).…”

Section: Discussionmentioning

confidence: 99%

“…Well known (direct or indirect) targets of PKA phosphorylation are 1) enzymes involved in trehalose synthesis and degradation, including trehalase and trehalose-6-phosphate synthase; 2) those involved in glycogen synthesis and degradation, such as glycogen synthase and glycogen phosphorylase; and 3) those enzymes involved in the glycolytic flux, such as phosphofructokinase and fructose-bisphosphatase (3,7). A less known target is pyruvate kinase, whose in vitro phosphorylation by PKA has recently been reported (8,9). The level of expression of PFK26 and PFK27 genes, coding for 6-phosphofructo-2-kinase, depend on the carbon source, via a cAMP pathway (10).…”

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confidence: 99%

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In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

Portela

Howell²,

Moreno

et al. 2002

Journal of Biological Chemistry

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Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HATpk1⅐Bcy1) 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 GSTPyk1 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 (tpk1 w1 ). 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 n H value of 1.4, as compared with an n H of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

Portela

Howell²,

Moreno

et al. 2002

Journal of Biological Chemistry

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“…PKA-dependent phosphorylation inhibits the activity of fructose-1,6-bisphosphatase (Fbp1) and stimulates the activity of 6-phosphofructo-2-kinase (Pfk2) and of both isoforms of pyruvate kinase (Pyk1 and Pyk2) (Gancedo et al 1983;Rittenhouse et al 1987;Cytrynska et al 2001;Vaseghi et al 2001;Portela et al 2002;Dihazi et al 2003). Together, these modifications result in the stimulation of glycolysis and the inhibition of gluconeogenesis when glucose is added to glucose-starved cells.…”

Section: Regulation Of the Camp-pka Pathwaymentioning

confidence: 99%

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

et al. 2010

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Cells of all living organisms contain complex signal transduction networks to ensure that a wide range of physiological properties are properly adapted to the environmental conditions. The fundamental concepts and individual building blocks of these signalling networks are generally well-conserved from yeast to man; yet, the central role that growth factors and hormones play in the regulation of signalling cascades in higher eukaryotes is executed by nutrients in yeast. Several nutrient-controlled pathways, which regulate cell growth and proliferation, metabolism and stress resistance, have been defined in yeast. These pathways are integrated into a signalling network, which ensures that yeast cells enter a quiescent, resting phase (G 0 ) to survive periods of nutrient scarceness and that they rapidly resume growth and cell proliferation when nutrient conditions become favourable again. A series of well-conserved nutrient-sensory protein kinases perform key roles in this signalling network: i.e. Snf1, PKA, Tor1 and Tor2, Sch9 and Pho85-Pho80. In this review, we provide a comprehensive overview on the current understanding of the signalling processes mediated via these kinases with a particular focus on how these individual pathways converge to signalling networks that ultimately ensure the dynamic translation of extracellular nutrient signals into appropriate physiological responses.

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“…Furthermore, PKA directly regulates important metabolic pathways such as glycolysis (6). It phosphorylates proteins catalyzing key enzymatic steps, such as both isoforms of pyruvate kinase, Cdc19 and Pyk2 (13)(14)(15).…”

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confidence: 99%

Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae

Nicastro

Tripodi

Gaggini

et al. 2015

Journal of Biological Chemistry

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Background:The Snf1/AMPK and PKA pathways are crucial for nutrient sensing and utilization in yeast. Results: A novel cross-talk mechanism between Snf1/AMPK and PKA is proposed. Conclusion: Snf1 and Cyr1 interact in a nutrient-independent manner. Active Snf1 phosphorylates Cyr1 and negatively regulates cAMP content and PKA-dependent transcription. Significance: This is the first evidence of regulation of PKA pathway by Snf1/AMPK.

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Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro

Cited by 8 publications

References 16 publications

In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae

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