Essential Residues for 14-3-3 ANAAT Stable Complex Formation

M.B. CARBONETTO, A.A. IGLESIAS, D.M. BUSTOS

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual de SAIB; 2007

SAIB

SB-P03.ESSENTIAL RESIDUES FOR 14-3-3 ANAAT STABLECOMPLEXFORMATIONCarbonetto MB, Iglesias AA, BustosDM.Lab Biología y Bioinformática Estructural, IIB-INTECH(CONICET), Cam Circ Lag Km 6 cc164, Chascomús. E-mail:mbelenc@fibertel.com.arin vivoThe importance of 14-3-3 protein family has lately risen to a keyposition in cell biology because these proteins contribute to a widerange of regulatory processes in the cell. These are small proteins(~30 kD) that form both homo- and heterodimers and can bindmore than 200 target proteins. These partner proteins are veryspecific and can bind 14-3-3 with higher affinity in aphosphorylation dependent manner. Therefore specificity andaffinity are concepts that require special care when regards to 14-3-3 complexes. To get further understanding of the h14-3-3/oANAAT complex we performed BimolecularFluorescence Complementation (BiFC) assay. For this purpose, 4different ANAAT mutants were created by site directedmutagenesis: glutamic acids 50 and 87, defined as anchor residues,and arginines 53 and 89, defined as hot spot, were replaced withalanines. Mutants were cloned in mammalian expression vectorsfused to truncatedYFP, as well as human wild type h14-3-3z. HeLacells were visualized 24 hours after transfection with theseconstructs. Results indicate that these residues are essential forstable complex formation. oANAAT anchor and hot spot mutantscould not interact in a stable manner with h14-3-3. The correlationbetween in silico predictions and in vivo observations, in cellularphysiological conditions, was corroborated.