Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit
Protein kinase A (PKA), in yeast, plays a major role in controlling metabolism and gene expression in connection with the available nutrient conditions. We here measure, for the first time, a transient change in the in vivo PKA activity, along a cAMP peak produced by 100 mM glucose addition to glyce...
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2006
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_08986568_v18_n7_p1072_Portela http://hdl.handle.net/20.500.12110/paper_08986568_v18_n7_p1072_Portela |
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paper:paper_08986568_v18_n7_p1072_Portela2023-06-08T15:49:26Z Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit Portela, Paula Moreno de Colonna, Silvia cAMP signalling Glucose In situ assay Phosphorylation PKA (protein kinase A) Yeast cyclic AMP dependent protein kinase G protein coupled receptor glucose glycerol animal cell article breathing catalysis cell membrane permeability cell metabolism down regulation extract gel electrophoresis in vivo study nonhuman priority journal protein phosphorylation Saccharomyces cerevisiae upregulation Catalytic Domain Cyclic AMP Cyclic AMP-Dependent Protein Kinases Enzyme Activation Glucose Glycerol Mutation Oligopeptides Phosphorylation Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Signal Transduction Animalia Saccharomyces cerevisiae Protein kinase A (PKA), in yeast, plays a major role in controlling metabolism and gene expression in connection with the available nutrient conditions. We here measure, for the first time, a transient change in the in vivo PKA activity, along a cAMP peak produced by 100 mM glucose addition to glycerol-growing cells as well as a change in the phosphorylation state of its catalytic subunit (Tpk1p) following PKA activation. PKA activity was measured in situ in permeabilized cells, preserving its intracellular localization. Comparison of total PKA activity, measured in situ in permeabilized cells with data obtained from in vitro assays in crude extracts, underscores the inhibitory potency of the regulatory subunit within the cell. Tpk1p phosphorylation was detected through non-denaturing gel electrophoresis. Phosphorylation of Tpk1p increases its specificity constant toward kemptide substrate. The use of mutants of the cAMP pathway showed that phosphorylation depends on the activation of PKA via the G-protein coupled receptor pathway triggered by glucose. The phosphorylation state of Tpk1p was followed during the diauxic shift. Tpk1p phosphorylation is dynamic and reversible: its up-regulation correlates with a fully fermentative metabolism, while its down-regulation with stationary phase or respiratory metabolism. Reversible phosphorylation can thus be considered a new control mechanism possibly pointing to a fine-tuning of PKA activity in response to environmental conditions. © 2005 Elsevier Inc. All rights reserved. Fil:Portela, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Moreno, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2006 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_08986568_v18_n7_p1072_Portela http://hdl.handle.net/20.500.12110/paper_08986568_v18_n7_p1072_Portela |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
cAMP signalling Glucose In situ assay Phosphorylation PKA (protein kinase A) Yeast cyclic AMP dependent protein kinase G protein coupled receptor glucose glycerol animal cell article breathing catalysis cell membrane permeability cell metabolism down regulation extract gel electrophoresis in vivo study nonhuman priority journal protein phosphorylation Saccharomyces cerevisiae upregulation Catalytic Domain Cyclic AMP Cyclic AMP-Dependent Protein Kinases Enzyme Activation Glucose Glycerol Mutation Oligopeptides Phosphorylation Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Signal Transduction Animalia Saccharomyces cerevisiae |
| spellingShingle |
cAMP signalling Glucose In situ assay Phosphorylation PKA (protein kinase A) Yeast cyclic AMP dependent protein kinase G protein coupled receptor glucose glycerol animal cell article breathing catalysis cell membrane permeability cell metabolism down regulation extract gel electrophoresis in vivo study nonhuman priority journal protein phosphorylation Saccharomyces cerevisiae upregulation Catalytic Domain Cyclic AMP Cyclic AMP-Dependent Protein Kinases Enzyme Activation Glucose Glycerol Mutation Oligopeptides Phosphorylation Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Signal Transduction Animalia Saccharomyces cerevisiae Portela, Paula Moreno de Colonna, Silvia Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit |
| topic_facet |
cAMP signalling Glucose In situ assay Phosphorylation PKA (protein kinase A) Yeast cyclic AMP dependent protein kinase G protein coupled receptor glucose glycerol animal cell article breathing catalysis cell membrane permeability cell metabolism down regulation extract gel electrophoresis in vivo study nonhuman priority journal protein phosphorylation Saccharomyces cerevisiae upregulation Catalytic Domain Cyclic AMP Cyclic AMP-Dependent Protein Kinases Enzyme Activation Glucose Glycerol Mutation Oligopeptides Phosphorylation Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Signal Transduction Animalia Saccharomyces cerevisiae |
| description |
Protein kinase A (PKA), in yeast, plays a major role in controlling metabolism and gene expression in connection with the available nutrient conditions. We here measure, for the first time, a transient change in the in vivo PKA activity, along a cAMP peak produced by 100 mM glucose addition to glycerol-growing cells as well as a change in the phosphorylation state of its catalytic subunit (Tpk1p) following PKA activation. PKA activity was measured in situ in permeabilized cells, preserving its intracellular localization. Comparison of total PKA activity, measured in situ in permeabilized cells with data obtained from in vitro assays in crude extracts, underscores the inhibitory potency of the regulatory subunit within the cell. Tpk1p phosphorylation was detected through non-denaturing gel electrophoresis. Phosphorylation of Tpk1p increases its specificity constant toward kemptide substrate. The use of mutants of the cAMP pathway showed that phosphorylation depends on the activation of PKA via the G-protein coupled receptor pathway triggered by glucose. The phosphorylation state of Tpk1p was followed during the diauxic shift. Tpk1p phosphorylation is dynamic and reversible: its up-regulation correlates with a fully fermentative metabolism, while its down-regulation with stationary phase or respiratory metabolism. Reversible phosphorylation can thus be considered a new control mechanism possibly pointing to a fine-tuning of PKA activity in response to environmental conditions. © 2005 Elsevier Inc. All rights reserved. |
| author |
Portela, Paula Moreno de Colonna, Silvia |
| author_facet |
Portela, Paula Moreno de Colonna, Silvia |
| author_sort |
Portela, Paula |
| title |
Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit |
| title_short |
Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit |
| title_full |
Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit |
| title_fullStr |
Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit |
| title_full_unstemmed |
Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit |
| title_sort |
glucose-dependent activation of protein kinase a activity in saccharomyces cerevisiae and phosphorylation of its tpk1 catalytic subunit |
| publishDate |
2006 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_08986568_v18_n7_p1072_Portela http://hdl.handle.net/20.500.12110/paper_08986568_v18_n7_p1072_Portela |
| work_keys_str_mv |
AT portelapaula glucosedependentactivationofproteinkinaseaactivityinsaccharomycescerevisiaeandphosphorylationofitstpk1catalyticsubunit AT morenodecolonnasilvia glucosedependentactivationofproteinkinaseaactivityinsaccharomycescerevisiaeandphosphorylationofitstpk1catalyticsubunit |
| _version_ |
1768546307562012672 |