14-3-3 Protein Interacts with Nuclear Localization Sequence of Forkhead Transcription Factor FoxO4

Abstract

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner. 14-3-3 proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of nuclear localization sequence (NLS). Masking or obscuring of NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equilibrium of FoxO localization toward the cytoplasm. According to our best knowledge, there is no experimental evidence showing a direct interaction between the 14-3-3 protein and NLS of FoxO. Therefore, the main goal of this work was to investigate whether the phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of FoxO4 NLS. We have used site-directed labeling of FoxO4 NLS with the extrinsic fluorophore 1,5-IAEDANS in conjunction with steady-state and time-resolved fluorescence spectroscopy to study conformational changes of FoxO4 NLS in vitro. Our data show that the 14-3-3 protein binding significantly changes the environment around AEDANS-labeled NLS and reduces its flexibility. On the other hand, the phosphorylation itself and the binding of double-stranded DNA have a small effect on the structure of this region. Our results also suggest that the DNA-binding domain of FoxO4 remains relatively mobile while bound to the 14-3-3 protein.

Structure/function relationships underlying regulation of FOXO transcription factors

“…Examples of this mode of regulation include protein phosphatase Cdc25C (Dalal et al, 1999), histone deacetylase (Grozinger and Schreiber, 2000), telomerase (Seimiya et al, 2000) and protein kinase U-a (Zhang et al, 1999). In agreement with this hypothesis, the 14-3-3 protein has been shown to physically interact with both parts of bipartite NLS of phosphorylated FOXO4 (fragment 11-213) (Obsilova et al, 2005).…”

“…Phosphorylation by AKT/PKB creates two binding sites for the 14-3-3 proteins and induces phosphorylation of additional sites by casein kinase-1 (CK1) and dual-specificity tyrosineregulated kinase-1A. The AKT/PKB-mediated phosphorylation of FOXO induces binding of 14-3-3 proteins, and the resulting complex is then translocated to the cytosol where the bound 14-3-3 protein prevents reentry of FOXO into the nucleus likely by interfering with the function of their NLS (Brunet et al, 1999;Brownawell et al, 2001;Cahill et al, 2001;Zhao et al, 2004;Obsilova et al, 2005). In addition to AKT/PKB-mediated phosphorylation, the function of FOXO proteins is also controlled by other types of posttranslational modifications including non-AKT/ PKB-mediated phosphorylation, acetylation and ubiquitination.…”

The FoxO code

“…The phosphorylation of FoxO by Akt and SGK at the first and second phosphorylation sites (T32 and S253 in FoxO3) creates binding sites for the chaperone protein 14-3-3 (Brunet et al, 1999;Obsilova et al, 2005;Rinner et al, 2007;Li et al, 2007a). 14-3-3 binds to FoxO factors in the nucleus and allows their active export, probably by helping expose FoxO nuclear export sequence (Figure 3).…”

“…14-3-3 binds to FoxO factors in the nucleus and allows their active export, probably by helping expose FoxO nuclear export sequence (Figure 3). The binding of 14-3-3 also affects the flexibility of FoxO nuclear localization signal (Obsilova et al, 2005), thereby further preventing FoxO re-entry into the nucleus (Figure 3). In addition, phosphorylation of the second site (S256 in FoxO1) prevents FoxO re-entry into the nucleus by introducing a negative charge in the basic stretch of residues that forms the nuclear localization signal (Figure 3) (Rena et al, 2001).…”

Post-translational modifications of FOXO family proteins

“…at Thr28 and Ser193 downregulates the transcription activity of the protein by suppressing DNA binding (22,25). In addition, Akt also induces the degradation of FOXO via the proteasomal pathway (26)(27)(28).…”