14-3-3 Protein Masks the DNA Binding Interface of Forkhead Transcription Factor FOXO4

Abstract

The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the FOXO4⅐14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4.The forkhead family of transcription factors shares a highly conserved 100-amino acid large DNA-binding domain (DBD) 2 or FOX (Forkhead box) domain. The FOX proteins display large functional diversity and play a wide range of roles in a number of physiological and pathological processes (1-3). Among the forkhead family, the FOXO class consists of four members (FOXO1, FOXO3, FOXO4, and FOXO6) that play a central role in cell cycle control, differentiation, metabolism control, stress response, and apoptosis (4 -6). Transcriptional activity of FOXO proteins is regulated through the insulinphosphatidylinositol 3-kinase-AKT/protein kinase B (PKB) signaling pathway. The AKT/PKB-mediated phosphorylation triggers phosphorylation of additional sites by casein kinase-1 and dual specificity tyrosine-regulated kinase-1A and induces FOXO binding to the 14-3-3 protein. This in turn both promotes the nuclear export of the resulting complex and inhibits the nuclear import of FOXO, probably by interfering with the function of its nuclear localization signal (NLS) (7-11). In addition to phosphorylation, the function of FOXO proteins is further controlled by other types of posttranslational modifications, including acetylation and ubiquitination (12, 13).The AKT/PKB-dependent phosphorylation of FOXO proteins generates two 14-3-3 binding sites. First motif is located close to the N terminus of FOXO molecule and the second one at t...

Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1

“…This could be attributed to a bound fraction of DANS-TRE sensing lower solvation and decreased microenvironmental polarity in the binding pocket of the complex. The value is consistent with values commonly found for the dansyl moiety covalently linked to globular proteins (34,35). The fractional intensity of the 14.3-ns component in the Bmh1:pNth1 1-751 (E674A) complex (4.2%) is significantly higher than in the presence of pNth1 1-751 (E674A) (0.5%) or Bmh1 alone (1.3%), where the presence of the long component likely indicates some nonspecific interaction of DANS-TRE with Bmh1.…”

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

“…These proteins contain a nuclear localization sequence whose interaction with the nuclear import machinery is inhibited upon the 14-3-3 protein binding. Recently, we have also shown that the forkhead transcription factor FOXO4 is inhibited by a similar mechanism involving the physical occlusion of its DNA binding interface (26).…”

“…Labeling of 14-3-3 Protein Mutants by 5-Iodoacetamidofluorescein for FRET Measurement-Covalent modification of the 14-3-3 protein mutants containing a single cysteine residue with the thiol-reactive probe 5-iodoacetamidofluorescein was carried out as described elsewhere (26,28). Briefly, we constructed two mutants containing a single cysteine residue (either at position 25 or at position 189) of human monomeric 14-3-3 protein (mutant S58D) (30).…”

“…Fluorescence Resonance Energy Transfer Analysis-The fluorescence resonance energy transfer data were measured and analyzed as described previously (26,28). The Förster critical distance, R 0 , was calculated as follows,…”

“…To prepare well defined pRGS3 and 14-3-3 complexes containing only one donor-acceptor pair of fluorophores, we used previously prepared versions of the monomeric form of 14-3-3 (mutation S58D) containing a single cysteine residue either at position 25 or at position 189, respectively (26,28,30). A mutant of pRGS3 containing a single cysteine residue within the RGS domain at position 456 was used to specifically label the RGS domain (Fig.…”