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 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.
Portela
Paula
P
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Piso 4, Buenos Aires 1428, Argentina.
Moreno
Silvia
S
D020134:Catalytic Domain; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005947:Glucose / Q000502:physiology*; D005990:Glycerol / Q000737:chemistry; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D009842:Oligopeptides; D029701:Saccharomyces cerevisiae Proteins; C037642:kemptide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose; D005990:Glycerol