14-3-3 Protein Masks the DNA Binding Interface of Forkhead Transcription Factor FOXO4
2009; Elsevier BV; Volume: 284; Issue: 29 Linguagem: Inglês
10.1074/jbc.m109.002725
ISSN1083-351X
AutoresJan Šilhán, Petr Vácha, Pavla Strnadova, Jaroslav Večeř, Petr Heřman, Miroslav Šulc, Jan Teisinger, Veronika Obšilová, Tomáš Obšil,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe 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 FOXO4·14-3-3 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 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 FOXO4·14-3-3 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) 2The abbreviations used are: DBDDNA-binding domain1,5-IAEDANS5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acidAEDANS5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acidPKBprotein kinase BdpFOXO4-(11–213)doubly phosphorylated FOXO4- (11–213)pFOXO4-(82–207)phosphorylated FOXO4-(82–207)FRETFörster resonance energy transferJNKc-Jun N-terminal kinaseMALDI-TOFmatrix-assisted laser desorption ionization-time of flightNLSnuclear localization signaldsDNAdouble-stranded DNA. 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.Weigel D. Jäckle H. Cell. 1990; 63: 455-456Abstract Full Text PDF PubMed Scopus (399) Google Scholar, 2.Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar, 3.Mazet F. Yu J.K. Liberles D.A. Holland L.Z. Shimeld S.M. Gene. 2003; 316: 79-89Crossref PubMed Scopus (94) Google Scholar). 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.Calnan D.R. Brunet A. Oncogene. 2008; 27: 2276-2288Crossref PubMed Scopus (936) Google Scholar, 5.Fu Z. Tindall D.J. Oncogene. 2008; 27: 2312-2319Crossref PubMed Scopus (449) Google Scholar, 6.Gross D.N. van den Heuvel A.P.J. Birnbaum M.J. Oncogene. 2008; 27: 2320-2336Crossref PubMed Scopus (433) Google Scholar). Transcriptional activity of FOXO proteins is regulated through the insulin-phosphatidylinositol 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.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 8.Brownawell A.M. Kops G.J. Macara I.G. Burgering B.M. Mol. Cell. Biol. 2001; 21: 3534-3546Crossref PubMed Scopus (269) Google Scholar, 9.Cahill C.M. Tzivion G. Nasrin N. Ogg S. Dore J. Ruvkun G. Alexander-Bridges M. J. Biol. Chem. 2001; 276: 13402-13410Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 10.Zhao X. Gan L. Pan H. Kan D. Majeski M. Adam S.A. Unterman T.G. Biochem. J. 2004; 378: 839-849Crossref PubMed Scopus (186) Google Scholar, 11.Obsilova V. Vecer J. Herman P. Pabianova A. Sulc M. Teisinger J. Boura E. Obsil T. Biochemistry. 2005; 44: 11608-11617Crossref PubMed Scopus (95) Google Scholar). In addition to phosphorylation, the function of FOXO proteins is further controlled by other types of posttranslational modifications, including acetylation and ubiquitination (12.van der Horst A. Burgering B.M.T. Nat. Rev. Mol. Cell Biol. 2007; 8: 440-450Crossref PubMed Scopus (595) Google Scholar, 13.Obsil T. Obsilova V. Oncogene. 2008; 27: 2263-2275Crossref PubMed Scopus (179) Google Scholar). DNA-binding domain 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid protein kinase B doubly phosphorylated FOXO4- (11–213) phosphorylated FOXO4-(82–207) Förster resonance energy transfer c-Jun N-terminal kinase matrix-assisted laser desorption ionization-time of flight nuclear localization signal double-stranded DNA. 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 the C terminus of DBD (Fig. 1A). It has been shown that simultaneous use of both 14-3-3 binding motifs is necessary for optimal FOXO binding to the 14-3-3 proteins (7.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 10.Zhao X. Gan L. Pan H. Kan D. Majeski M. Adam S.A. Unterman T.G. Biochem. J. 2004; 378: 839-849Crossref PubMed Scopus (186) Google Scholar, 14.Obsil T. Ghirlando R. Anderson D.E. Hickman A.B. Dyda F. Biochemistry. 2003; 42: 15264-15272Crossref PubMed Scopus (84) Google Scholar). These two motifs border the DBD, raising the possibility that the 14-3-3 proteins could participate in the disruption of FOXO binding to the DNA. Such 14-3-3 protein-dependent inhibition of DNA binding has been suggested for DAF-16 (Caenorhabditis elegans FOXO homologue) and FOXO4 (9.Cahill C.M. Tzivion G. Nasrin N. Ogg S. Dore J. Ruvkun G. Alexander-Bridges M. J. Biol. Chem. 2001; 276: 13402-13410Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 14.Obsil T. Ghirlando R. Anderson D.E. Hickman A.B. Dyda F. Biochemistry. 2003; 42: 15264-15272Crossref PubMed Scopus (84) Google Scholar). However, the exact mechanism of this 14-3-3-dependent inhibition of FOXO binding to DNA is still unclear. Since the second AKT/PKB motif is embedded in the C-terminal part of DBD, also known as the wing W2, the 14-3-3 protein can interfere with the binding of this region to DNA, mask other parts of the FOXO DNA binding interface, or change the conformation of the forkhead domain core. To better understand the mechanism of 14-3-3-dependent regulation of FOXO function, we investigated which regions of FOXO4 DBD, apart from the C-terminal phosphorylated motif, physically interact with 14-3-3ζ protein. We labeled four different sites within the FOXO4 DBD (N-terminal region, C-terminal wing W2, and both sides of helix H3) with the extrinsic fluorophore 1,5-IAEDANS and used methods of time-resolved fluorescence spectroscopy to study the interaction between FOXO4 and the 14-3-3ζ protein. Our results suggest that the 14-3-3ζ protein physically interacts with all four tested regions of DBD that represent important parts of its DNA binding interface. Such interactions probably mask the DNA binding interface, thus blocking the FOXO4 binding to the target DNA. The 14-3-3ζ 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 FOXO4·14-3-3ζ complex, we measured six distances between 14-3-3ζ and FOXO4 using Förster resonance energy transfer (FRET) 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 the 14-3-3 protein masks the DNA binding interface of FOXO4. All mutants of FOXO4 (sequences 11–213 and 82–207) were generated using the QuikChange kit (Stratagene). All mutations were confirmed by sequencing. Both wild type and all mutants of FOXO4-(11–213) and FOXO4-(82–207) were expressed, purified, and phosphorylated as described previously (11.Obsilova V. Vecer J. Herman P. Pabianova A. Sulc M. Teisinger J. Boura E. Obsil T. Biochemistry. 2005; 44: 11608-11617Crossref PubMed Scopus (95) Google Scholar, 15.Boura E. Silhan J. Herman P. Vecer J. Sulc M. Teisinger J. Obsilova V. Obsil T. J. Biol. Chem. 2007; 282: 8265-8275Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The completeness of the phosphorylation reaction was checked using MALDI-TOF mass spectrometry. Samples were first separated by 12% SDS-PAGE, and excised protein bands were digested with trypsin endoprotease (Promega) directly in gel with cysteine modification by iodoacetamide (16.Sadilkova L. Osicka R. Sulc M. Linhartova I. Novak P. Sebo P. Protein Sci. 2008; 17: 1834-1843Crossref PubMed Scopus (30) Google Scholar). The resulting peptide mixtures were extracted and loaded on a MALDI-TOF target with α-cyano-4-hydroxy-cinnamic acid as matrix, and positively/negatively charged spectra were acquired on a BIFLEX mass spectrometer (Bruker-Daltonics, Bremen, Germany) with internal calibration using the monoisotopic [M + H]+/[M − H]− ions of the FOXO4 peptides with known sequence, as described previously (11.Obsilova V. Vecer J. Herman P. Pabianova A. Sulc M. Teisinger J. Boura E. Obsil T. Biochemistry. 2005; 44: 11608-11617Crossref PubMed Scopus (95) Google Scholar). Covalent modification of FOXO4-(11–213) and FOXO4-(82–207) containing a single cysteine residue with the thiol-reactive probe 1,5-IAEDANS was carried out as described elsewhere (15.Boura E. Silhan J. Herman P. Vecer J. Sulc M. Teisinger J. Obsilova V. Obsil T. J. Biol. Chem. 2007; 282: 8265-8275Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 17.Silhan J. Obsilova V. Vecer J. Herman P. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 49113-49119Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, the protein (50–70 μm) in 50 mm Tris (pH 7.5), 100 mm NaCl, 1 mm EDTA, 10% (v/w) glycerol, and label were mixed at a molar ratio of 1:40 and incubated at 30 °C for 2 h and then at 4 °C overnight in the dark. The free unreacted label was removed by size exclusion chromatography on a Superdex 75 10/300 GL column in a buffer containing 50 mm Tris (pH 7.5), 100 mm NaCl, 1 mm EDTA, and 10% (v/w) glycerol. The incorporation stoichiometry was determined by comparing the peak protein absorbance at 280 nm with the absorbance of bound AEDANS measured at 336 nm using the extinction coefficient of 5,700 m−1 cm−1 (Molecular Probes, Inc., Eugene, OR). Human 14-3-3 protein (ζ-isoform) was expressed and purified as described previously (18.Obsil T. Ghirlando R. Klein D.C. Ganguly S. Dyda F. Cell. 2001; 105: 257-267Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). To prepare the 14-3-3 protein suitable for FRET measurements, we constructed two mutants containing a single cysteine residue (either at position 25 or 189) of human monomeric 14-3-3ζ protein (mutant S58D) (19.Woodcock J.M. Murphy J. Stomski F.C. Berndt M.C. Lopez A.F. J. Biol. Chem. 2003; 278: 36323-36327Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The protein (40 μm) in 50 mm Tris (pH 7.5), 100 mm NaCl, 1 mm EDTA, and label 5-iodoacetamidofluorescein (Invitrogen) were mixed at a molar ratio of 1:40 and incubated at room temperature for 8 h and then at 4 °C overnight in the dark. The free unreacted label was removed by dialysis and size exclusion chromatography. The incorporation stoichiometry was determined by the absorbance at 492 nm using an extinction coefficient of 78,000 m−1 cm−1 (Molecular Probes). The stoichiometry of fluorescein incorporation/mol of protein was found to be 99–100%. Steady-state fluorescence measurements were performed on a PerkinElmer Life Sciences LS50B fluorescence spectrometer at 17 °C in a buffer containing 50 mm Tris (pH 7.5), 100 mm NaCl, and 1 mm EDTA with 100 nm double-stranded DNA (dsDNA) labeled with fluorescein at the 5′ terminus of one strand. The following oligonucleotides containing the consensus FOXO binding sequence were used to prepare the samples of dsDNA: 5′-fluorescein-GCGTTGTTTACGC-3′ and 5′-GCGTAAACAACGC-3′ (15.Boura E. Silhan J. Herman P. Vecer J. Sulc M. Teisinger J. Obsilova V. Obsil T. J. Biol. Chem. 2007; 282: 8265-8275Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Increasing amounts of the protein were titrated into the cuvette. At each FOXO4-(11–213) concentration, the steady-state fluorescence anisotropy of fluorescein was recorded (excitation at 494 nm and emission at 520 nm). Anisotropy was calculated from the fluorescence intensities according to the relationship, r = (I‖ − I⊥)/(I‖ + 2I⊥). The fraction of the DNA bound (FB) was calculated from the formula,FB=robs−rmin/rmax−robsQ+robs−rmin(Eq. 1) where Q represents the quantum yield ratio of the bound to the free form, and it was estimated by the ratio of the intensities of the bound to the free fluorophore. Parameter rmax is the anisotropy at saturation, robs is the observed anisotropy for any FOXO4-(11–213) concentration, and rmin is the minimum observed anisotropy of the free DNA. FB was plotted against the FOXO4-(11–213) protein concentration and fitted using Equation 2 (20.Kohler J.J. Schepartz A. Biochemistry. 2001; 40: 130-142Crossref PubMed Scopus (99) Google Scholar) to determine KD for the FOXO4-(11–213)·dsDNA complex formation,FB=KD+P1+P2−KD+P1+P22−4P1P22P1(Eq. 2) where KD is the equilibrium dissociation constant, [P1] is the dsDNA-fluorescein concentration, and [P2] is the FOXO4-(11–213) concentration. Nonlinear data fitting was performed using the Origin version 6.0 package (Microcal Software Inc.). Steady-state fluorescence measurements were performed with 0.5 μm FOXO4-(11–213) labeled with 1,5-IAEDANS in a buffer containing 50 mm Tris (pH 7.5), 100 mm NaCl, and 1 mm EDTA. Increasing amounts of the 14-3-3ζ protein were titrated into the cuvette. At each 14-3-3ζ concentration, the steady-state fluorescence anisotropy of AEDANS was measured (excitation at 336 nm and emission at 490 nm). Data were analyzed as described above in the case of the DNA-binding studies. Fluorescence intensity and anisotropy decays were measured on a time-correlated single photon counting apparatus, as described previously (17.Silhan J. Obsilova V. Vecer J. Herman P. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 49113-49119Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 21.Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The fluorescence decays have been acquired under the "magic angle" conditions when the measured intensity decay, I(t), is independent of the rotational diffusion of the chromophore and provides unbiased information about lifetimes. The apparatus response function was measured with a diluted Ludox (colloidal silica) solution. Samples were placed in a thermostatic holder, and all experiments were performed at 22 °C in a buffer containing 50 mm Tris-HCl (pH 7.5), 100 mm NaCl, and 1 mm EDTA. The FOXO4-(11–213) concentration was 15 μm; the 14-3-3 protein concentration was 30 μm. Dansyl fluorescence was excited at 315 nm by an actively mode-locked picosecond dye laser (Spectra Physics). Fluorescence was collected at 480 nm using a monochromator with a 400 nm cut-off filter placed in front of its input slit. Tryptophan emission was excited at 297 nm by a pulsed UV-LED with a 500-ps pulse width (PLS 295-10; PicoQuant). Tryptophan emission was collected at 355 nm through a monochromator complemented by a UG1 glass filter (Zeiss) in front of the input slit. The fluorescence decays were processed as described previously (17.Silhan J. Obsilova V. Vecer J. Herman P. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 49113-49119Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 21.Obsilova V. Herman P. Vecer J. Sulc M. Teisinger J. Obsil T. J. Biol. Chem. 2004; 279: 4531-4540Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), using the singular value decomposition maximum entropy method (22.Bryan R.K. Eur. Biophys. J. 1990; 18: 165-174Crossref Scopus (259) Google Scholar). Fluorescence was assumed to decay multiexponentially according to the formula,It=∑iαi⋅e−t/τi(Eq. 3) where τi and αi are the fluorescence lifetimes and the corresponding amplitudes, respectively. I(t) was analyzed by the singular value decomposition maximum entropy method coded according to Bryan (22.Bryan R.K. Eur. Biophys. J. 1990; 18: 165-174Crossref Scopus (259) Google Scholar). The program yields a set of amplitudes αi that represents the lifetime distribution in the decay. We have chosen 100 lifetimes covering the range from 20 ps to 20 ns. The lifetimes were equidistantly spaced in the logarithmic scale. Not having prior information about the number of lifetime classes (peaks) in the lifetime distribution and about the distribution shape, the data analysis started from the flat initial guess, where all lifetimes have the same prior probability. The mean lifetime τ̄ was calculated as follows,τ¯=∑ifiτi(Eq. 4) where fi represents the fractional intensity of the ith lifetime component.fi=αiτi/∑iαiτi(Eq. 5) The fluorescence anisotropy decays, r(t), were obtained from the parallel I‖(t) and perpendicular I⊥(t) decay components. Data were analyzed by a method similar to the one published by Brochon (23.Brochon J.C. Methods Enzymol. 1994; 240: 262-311Crossref PubMed Scopus (254) Google Scholar), using a program developed at the Institute of Physics, Charles University (Prague, Czech Republic). We used a model-independent singular value decomposition maximum entropy method approach that does not set prior limits on the shape of the distribution. The anisotropies were analyzed for a series of exponentials,rt=∑iβi⋅e−t/ϕi(Eq. 6) where the amplitudes βi represent the distribution of the correlation times ϕi. βi values are related to the initial anisotropy r0 by the formula,∑βi=r0(Eq. 7) We used 100 correlation times ϕi equidistantly spaced in the logarithmic scale and ranging from 100 ps to 500 ns. AEDANS fluorescence was quenched by acrylamide. The fluorescence decays were collected at 480 nm in the presence of different quencher concentrations [Q]. The time-resolved quenching data were analyzed by the Stern-Volmer equation,τ¯0/τ¯=1+KSV⋅Q=1+kqτ¯0⋅Q(Eq. 8) where τ̄0 and τ̄ represent the mean fluorescence lifetime in the absence and in the presence of acrylamide, respectively. Ksv and kq represent the phenomenological Stern-Volmer constant and the bimolecular quenching constant that reflects the collisional quenching efficiency of AEDANS by the quencher, respectively. The average efficiency of fluorescence energy transfer E was calculated from the mean donor (AEDANS) lifetime in the presence (τDA) and absence of the acceptor (τD).E=1−τDAτD(Eq. 9) The average distance between the donor-acceptor pair R was calculated from Equation 10,R=R06E−1−1(Eq. 10) where R0 is the Förster critical distance. R0 is given by Ref. 24.Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 305-315Google Scholar,R0=8.8×1023κ2n−4QDJλ1/6Å(Eq. 11) where n is the refractive index of the medium, QD is the emission quantum yield of the donor in the absence of the acceptor, and κ2 is the orientation factor. The spectral overlap integral J(λ) of donor fluorescence and acceptor absorption was calculated from Equation 12 (24.Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 305-315Google Scholar),Jλ=∫FDλεaλλ4dλ/∫FDλdλ cm3 M−1(Eq. 12) where FD(λ) and ϵa(λ) represent the fluorescence intensity of the donor and the molar extinction coefficient of the acceptor, respectively, at the wavelength λ expressed in cm. The orientation factor κ2 was assumed to be equal to ⅔, which corresponds to randomly oriented dipole moments of the donor-acceptor pairs. The value of 1.31 was used for the refractive index of the aqueous solution. The quantum yields of the donor, which is the AEDANS moiety attached at three different positions within the pFOXO4-(82–207) molecule, were calculated relative to the QD value of quinine sulfate in 0.1 m H2SO4 (25.Melhuish W.H. J. Phys. Chem. 1961; 65: 229-235Crossref Scopus (1653) Google Scholar). The main goal of this work was to characterize interactions between the 14-3-3ζ protein and the forkhead domain of transcription factor FOXO4 and, above all, to answer the question of how the 14-3-3 protein inhibits FOXO4 binding to the target DNA. The AKT/PKB-induced phosphorylation of FOXO factors, followed by their association with the 14-3-3 proteins, takes place in the nucleus. This interaction in turn inhibits the FOXO binding to DNA and promotes nuclear export of the resulting complex (7.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 8.Brownawell A.M. Kops G.J. Macara I.G. Burgering B.M. Mol. Cell. Biol. 2001; 21: 3534-3546Crossref PubMed Scopus (269) Google Scholar, 9.Cahill C.M. Tzivion G. Nasrin N. Ogg S. Dore J. Ruvkun G. Alexander-Bridges M. J. Biol. Chem. 2001; 276: 13402-13410Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 10.Zhao X. Gan L. Pan H. Kan D. Majeski M. Adam S.A. Unterman T.G. Biochem. J. 2004; 378: 839-849Crossref PubMed Scopus (186) Google Scholar, 11.Obsilova V. Vecer J. Herman P. Pabianova A. Sulc M. Teisinger J. Boura E. Obsil T. Biochemistry. 2005; 44: 11608-11617Crossref PubMed Scopus (95) Google Scholar). Since the 14-3-3 protein binding motifs border the DBD, it is reasonable to speculate that the 14-3-3 protein can either sterically block the DNA binding interface or change its structure. To test the former possibility, we prepared four mutants of FOXO4-(11–213) (human FOXO4, sequence 11–213) suitable for site-specific labeling of DBD with thiol-reactive fluorescence dye. The sequence of FOXO4-(11–213) covers the N-terminal half of the FOXO4 protein and contains the DBD flanked by two AKT/PKB phosphorylation/14-3-3 protein binding sites and NLS (Fig. 1A) (11.Obsilova V. Vecer J. Herman P. Pabianova A. Sulc M. Teisinger J. Boura E. Obsil T. Biochemistry. 2005; 44: 11608-11617Crossref PubMed Scopus (95) Google Scholar, 14.Obsil T. Ghirlando R. Anderson D.E. Hickman A.B. Dyda F. Biochemistry. 2003; 42: 15264-15272Crossref PubMed Scopus (84) Google Scholar). The amino acid sequence of FOXO4-(11–213) contains only one cysteine residue at position 27. To specifically label the selected sites within the FOXO4 DBD with a fluorescence probe, we mutated Cys27 to Ala and introduced new Cys residues at four different positions within the DBD. The sequence of DBD does not contain any cysteine residue; thus, serines located at four different positions were replaced with cysteines (Cys92, Cys142, Cys155, and Cys183) (Fig. 1B). Next, the fluorescence anisotropy-based binding assay was used to check the DNA-binding affinity of FOXO4-(11–213) single-Cys mutants. These experiments showed that FOXO4-(11–213) single-Cys mutants bind dsDNA, containing consensus FOXO binding sequence, with KD ranging from 28 to 42 nm (Fig. 2A). This is fully comparable with the DNA-binding affinity of FOXO4-(11–213) wild type, which binds DNA with a KD value of 34 ± 6 nm. The single-Cys mutants of FOXO4-(11–213) were then labeled with the extrinsic fluorophore 1,5-IAEDANS, and the stoichiometry of AEDANS incorporation/mol of the protein was found to be 96–100%. The AEDANS-labeled mutants of FOXO4-(11–213) were phosphorylated using AKT/PKB, which is known to stoichiometrically phosphorylate FOXO4-(11–213) in vitro at two sites, Thr28 and Ser193 (11.Obsilova V. Vecer J. Herman P. Pabianova A. Sulc M. Teisinger J. Boura E. Obsil T. Biochemistry. 2005; 44: 11608-11617Crossref PubMed Scopus (95) Google Scholar, 14.Obsil T. Ghirlando R. Anderson D.E. Hickman A.B. Dyda F. Biochemistry. 2003; 42: 15264-15272Crossref PubMed Scopus (84) Google Scholar). Both the modification by AEDANS and the completeness of phosphorylation were confirmed by MALDI-TOF mass spectrometry. The comparison of the mass spectra of digested sample with theoretically generated tryptic peptides clearly demonstrated the presence of two phosphorylated peptides having a protonated mass of 3163.4 (m/z), corresponding to the phosphorylated peptide SApTWPLPRPEIANQPSEPPEVEPDLGEK, and 1345.6 (m/z), matched to the phosphorylated peptide AApSMDSSSKLLR (where pS and pT denote phosphorylated serine and threonine residues, respectively). The tandem mass spectrometry fragmentation in postsource decay experiment confirmed the structure and phosphorylated amino acid of these peptides. The negative MALDI-TOF mass spectra of digested samples were also used to corroborate an AEDANS moiety at the cysteine residue of all single cysteine constructs. As one example of these results, the signal at 1983.9 (m/z) of the modified peptide SSWWMLNPEGGKC*GK by AEDANS was observed (where C* denotes a modified cysteine residue). On the other hand, the nonphosphorylated or unmodified protein mass spectra provided no peaks with the same values of m/z there; only the signals corresponding to the nonphosporylated or unmodified peptides were detected (data not shown). A fluorescence anisotropy-based binding assay was used to check the binding of the 14-3-3ζ protein to the AEDANS-labeled phosphorylated mutants of FOXO4-(11–213) (Fig. 2B). These experiments confirmed that the replacement of serines with cysteines and subsequent modification by 1,5-IAEDANS do not affect the FOXO4-(11–213) interaction with the 14-3-3ζ protein. The analysis of the binding curves yielded dissociation constants ranging from 60 to 120 nm (considering the FOXO4-(11–213)·14-3-3 complex with the 1:2 molar stoichiometry) (11.Obsilova V. Vecer J. Herman P. Pabianova A. Sulc M. Teisinger J. Boura E. Obsil T. Biochemistry. 2005; 44: 11608-11617Crossref PubMed Scopus (95) Google Scholar, 14.Obsil T. Ghirlando R. Anderson D.E. Hickman A.B. Dyda F. Biochemistry. 2003; 42: 15264-15272Crossref PubMed Scopus (84) Google Scholar). One possible mechanism of the 14-3-3 protein-dependent inhibition of the FOXO binding to the target DNA is the steric occlusion of the DNA binding interface by the 14-3-3 protein. To map the interaction of FOXO4 DBD with the 14-3-3ζ protein, time-resolved fluorescence intensity and anisotropy decay measurements of all four AEDANS-Cys mutants of FOXO4-(11–213) were performed. Since AEDANS is an environmentally sensitive fluorophore that changes the fluorescence lifetime according to its local interactions, a variation of the mean excited state lifetime, τ̄, sensitively reflects changes in polarity around the AEDANS moiety induced by the binding of the 14-3-3ζ protein. We have found that all AEDANS-labeled FOXO4-(11–213) mutants exhibited complex emission decays with multimodal lifetime distributions (e.g. see Fig. 3). The results of the emission decay analysis for all mutants are summarized in Table 1. It is seen that although AKT/PKB-induced phosphorylation itself has only a marginal effect on τ̄, the mean lifetime of all phosphorylated dpFOXO4-(11–213) mutants significantly increases upon the binding of the 14-3-3ζ protein. Si
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