In Vivo Phosphorylation of SER21 and SER83 during Nutrient-induced Activation of the Yeast PKA Target Trehalase.

Re-addition 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- to 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 Ser21 and Ser83. Unexpectedly, mutants with reduced PKA activity show constitutive phosphorylation in spite of reduced trehalase activation. The same phenotype was observed upon deletion of the catalytic subunits of yeast PP2A, 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 Bmh1,2 suggesting Dcs1 inhibits by preventing 14-3-3 binding. Deletion of Bmh1,2 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.
J. Biol. Chem.
Date: Nov. 15, 2012
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