title: In vivo phosphorylation of Ser21 and Ser83 during nutrient-induced activation of the yeast protein kinase A (PKA) target trehalase.
abstract: The readdition of an essential nutrient to starved, fermenting cells of the yeast Saccharomyces cerevisiae triggers rapid activation of the protein kinase A (PKA) pathway. Trehalase is activated 5-10-fold within minutes and has been used as a convenient reporter for rapid activation of PKA in vivo. Although trehalase can be phosphorylated and activated by PKA in vitro, demonstration of phosphorylation during nutrient activation in vivo has been lacking. We now show, using phosphospecific antibodies, that glucose and nitrogen activation of trehalase in vivo is associated with phosphorylation of Ser(21) and Ser(83). Unexpectedly, mutants with reduced PKA activity show constitutive phosphorylation despite reduced trehalase activation. The same phenotype was observed upon deletion of the catalytic subunits of yeast protein phosphatase 2A, suggesting that lower PKA activity causes reduced trehalase dephosphorylation. Hence, phosphorylation of trehalase in vivo is not sufficient for activation. Deletion of the inhibitor Dcs1 causes constitutive trehalase activation and phosphorylation. It also enhances binding of trehalase to the 14-3-3 proteins Bmh1 and Bmh2, suggesting that Dcs1 inhibits by preventing 14-3-3 binding. Deletion of Bmh1 and Bmh2 eliminates both trehalase activation and phosphorylation. Our results reveal that trehalase activation in vivo is associated with phosphorylation of typical PKA sites and thus establish the enzyme as a reliable read-out for nutrient activation of PKA in vivo.
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Schepers
authors.forename: Griet
authors.initials: G
authors.lastname: Van Zeebroeck
authors.forename: Martijn
authors.initials: M
authors.lastname: Pinkse
authors.forename: Peter
authors.initials: P
authors.lastname: Verhaert
authors.forename: Johan M
authors.initials: JM
authors.lastname: Thevelein
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014198:Trehalase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D029701:Saccharomyces cerevisiae Proteins; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; C457179:TPK2 protein, S cerevisiae; C579078:Tpk1 protein, S cerevisiae; C509333:Tpk3 protein, S cerevisiae; D014198:Trehalase; C478395:DCS1 protein, S cerevisiae; D009699:N-Glycosyl Hydrolases; D005947:Glucose; D009584:Nitrogen