Role of individual phosphorylation sites for the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1

Eva Macakova, Dana Veisova, Tomas Obsil and Veronika Obsilova

Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220 Prague, Czech Republic,

evamacakova@seznam.cz

Trehalases are important highly conserved enzymes found in a wide variety of organisms and responsible for the hydrolysis of trehalose to two glucose molecules. Trehalose, a naturally occurring nonreducing disaccharide, serves as a carbon and energy source as well as universal protectant from various stress conditions like dehydration, temperature extremes, oxidative stress and desiccation in a wide variety of organisms ranging from bacteria to invertebrates and higher plants. In yeast and plants it may also serve as a regulatory or signaling molecule to control certain metabolic pathways or even to affect growth.

Recently it has been shown that the enzymatic activity of neutral trehalase (Nth1) in yeast is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner through unknown mechanism. In this work, we investigated in detail the interaction between Saccharomyces cerevisiae Nth1 and 14-3-3 protein isoforms Bmh1 and Bmh2. The mass spectrometric analysis revealed that four residues within the disordered N-terminal segment of recombinant full length Nth1 (Ser20, Ser21, Ser60 and Ser83) are phosphorylated by PKA in vitro. Sedimentation analysis and enzyme kinetics measurements show that both yeast 14-3-3 isoforms form a stable complex with phosphorylated Nth1 and significantly enhance its enzymatic activity. The 14-3-3-dependent activation of Nth1 is significantly more potent compared to calcium-dependent activation. Limited proteolysis confirmed that 14-3-3 proteins interact with the N-terminal segment of Nth1 where all phosphorylation sites are located. Site-directed mutagenesis was used to decipher the importance of found phosphorylation sites for Nth1 activation.

In conclusion, our results show that S. cerevisiae Nth1 is phosphorylated by PKA at multiple sites out of which Ser60 and Ser83 are sites primarily responsible for PKA-dependent and 14-3-3-mediated activation of Nth1. Finally H/D exchange and cross-linking experiments coupled to mass spectrometry were used to determine the interacting surface of Nth1:Bmh1.

1. Veisova D, Macakova E, Rezabkova L, Sulc M, Vacha P, Sychrova H, Obsil T, Obsilova V., Biochemical Journal (2012) Feb 9 accepted DOI:10.1042/BJ20111615.

2. Veisova D, Rezabkova L, Stepanek M, Novotna P, Herman P, Vecer J, Obsil T, Obsilova V.: Biochemistry 49(2010): 3853 - 3861.

Financial support from Grant P207/11/0455 of the Czech Science Foundation, Grant 350111 of the Grant Agency of Charles University and Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic is gratefully acknowledged.