Visualization of 14-3-3 AANAT complex by bimolecular fluorescence complementation.

M BELÉN CARBONETTO, MARINA UHART, ALBERTO A IGLESIAS AND DIEGO M BUSTOS

Rosario

Congreso; Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular; 2006

SAIB

Serine and threonine kinases constitute ~92% of all kinases in humans. 14-3-3 proteins are novel members of this process. We report the implementation of bimolecular fluorescence complementation for the visualization of the 14-3-3 and serotonin N-acetyl transferase (AANAT) complex. In the later, E50 and E87 were defined as anchor residues, whereas R53 and R89 are hot spots by several in silico techniques. We compared 37 AANAT sequences to study conservation of these residues. Both anchor residues were strictly conserved in mammals, while only E50 was invariable in non-mammalian species. The hot spot R53 was strictly conserved in mammals, but R53 and R89 were variable in other species. Data support that anchors and hot spots are important in the 14-3-3-AANAT complex, which is only present in mammals. The interacting proteins were fused to sequences encoding Yellow Fluorescence Protein (YFP) fragments truncated at the residue 155, and cloned within mammalian expression vectors. Standard 17 aa linker residues were used, and the peptides Flag and HA, were maintained. After co-transfection with these constructs, HeLa cells were incubated 24 to 48 h and the stable association was visualized by the YFP fluorescence. We observed fluorescence in the cytoplasm only, due to the presence of a NES sequence in 14-3-3. Anchor and hot spot mutants complemented the analysis by this technique.