Role of individual phosphorylation sites for the 14-3-3-protein-dependent activation of yeast neutral trehalase Nth1.
Trehalases are important highly conserved enzymes found in a wide variety of organisms and are responsible for the hydrolysis of trehalose that serves as a carbon and energy source as well as a universal stress protectant. Emerging evidence indicates that the enzymatic activity of the neutral trehalase Nth1 in yeast is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner through an unknown mechanism. In the present study, we investigated in detail the interaction between Saccharomyces cerevisiae Nth1 and 14-3-3 protein isoforms Bmh1 and Bmh2. We determined four residues that are phosphorylated by PKA (protein kinase A) in vitro within the disordered N-terminal segment of Nth1. 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 with Ca2+-dependent activation. Limited proteolysis confirmed that the 14-3-3 proteins interact with the N-terminal segment of Nth1 where all phosphorylation sites are located. Site-directed mutagenesis in conjunction with the enzyme activity measurements in vitro and the activation studies of mutant forms in vivo suggest that Ser60 and Ser83 are sites primarily responsible for PKA-dependent and 14-3-3-mediated activation of Nth1.
Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220 Prague 4, Czech Republic.
Dana
D
Veisova
Eva
E
Macakova
Lenka
L
Rezabkova
Miroslav
M
Sulc
Petr
P
Vacha
Hana
H
Sychrova
Tomas
T
Obsil
Veronika
V
Obsilova
D048948:14-3-3 Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D009154:Mutation; D010766:Phosphorylation; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014198:Trehalase / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D014198:Trehalase