14-3-3 proteins bind to phosphorylated AANAT. (A) Two ³⁵S-labeled proteins bind to p-AANAT. ³⁵S-labeled cytosolic proteins were applied on GST-oAANAT (C) and GST-p-oAANAT (P) as described in Materials and Methods. The 30- and 33-kDa bands (arrow) bound preferentially to p-AANAT. (B) Mass spectrometric analysis of the two ³⁵S-labeled proteins that selectively bind to p-oAANAT. A representative full-scale MS/MS spectrum of the doubly charged ion at m/z 800.2 is presented, which was determined to result from the rat 14-3-3-β tryptic peptide, (K)AVTEQGHELSNEER. Observed y and b ions, including a continuous y₂-y₁₂ and b₇-b₁₃ series, consistent with those predicted for the peptide are labeled. Eleven other 14-3-3-derived peptides were identified, (K)AASDIAMTELPPTHPIR, (R)YLAEFATGNDRK, ɛ; (R)YLAEVAAGDDKK, (R)VVSSIEQK, (K)SVTEQGALELSNEER, ζ; (R)ATVVESSEK, (K)AYSEAHEISK, γ; (K)NVVGAR, β, γ, or ζ; (K)LAEQAER, (R)NLLSVAYK, β, γ, θ, or ζ; and (K)EMQPTHPIR, β, θ, or ζ. (C) Immunochemical identification of the bound proteins as 14-3-3 proteins. ³⁵S-labeled proteins were detected by autoradiograph (24 h) (Left) and immunodetected with antiserum As 274 (Center), which detects multiple 14-3-3 isoforms. Monoclonal antiserum As 8C3 (Right) detects 14-3-3-ɛ (33 kDa). The P/C ratios for radioactivity and immunoreactivity are >10. (D) HA-rAANAT coimmunoprecipitation of 14-3-3 in dibutyryl cAMP-treated C6 cells transfected with HA-rAANAT. Anti-HA was used to coimmunoprecipitate 14-3-3 from HA-rAANAT-transfected C6 cells, as described in Materials and Methods. IP antiserum As 274 was used to detect 14-3-3 proteins, as described in C.

N-terminal PK-A/14-3-3 site in AANAT is involved in 14-3-3 binding. (A) AANAT from ovine pineal gland coeluting with 14-3-3 is phosphorylated. Ovine pineal tissue (3̃00 mg; ref. 4) was homogenized in 3 ml of 0.1 M ammonium acetate buffer (pH 6.8; 4°C) and centrifuged (14,000 × g; 10 min). Proteins in 2.5 ml of the supernatant were fractionated by gel filtration [200 ml of TSK3000 column, Toya Soda, Tokyo; 0.1 M ammonium acetate buffer, pH 6.8, containing 10 mM DTT and 10% (vol/vol) glycerol; 1 ml/min flow rate; 4-ml fractions; 4°C]. AANAT activity was measured with 50 μl each 4-ml fraction (4). oAANAT was detected by Western blot with IP antisera As 2345 (1:125; raised against oAANAT_24–39) and with As 3352 (1:125; raised against p-rAANAT _22–37; ref. 4). The 14-3-3 proteins were detected with antiserum As 274. (B) Selectivity of antisera used. Western blot analysis of p-oAANAT and unphosphorylated oAANAT, using IP antisera As 2345 and 3352. IP antiserum As 2345 detects both phosphorylated and unphosphorylated forms; antiserum As 3352 detects only phosphorylated AANAT. (C) Coelution of hAANAT and 14-3-3 proteins in forskolin-treated cells expressing hAANAT. HA6 cells (see Materials and Methods) were either untreated (□—□) or treated (●—●) with forskolin (10 μM; 6 h), which elevated cAMP production >20-fold (data not presented). Chromatography and enzyme assays were as described in A. The 14-3-3 proteins were detected on a Western blot as in A and hAANAT was detected by using IP antiserum As 3236 raised against hAANAT_1–26. (D) p-DK14 (p-oAANAT_24–39) displaces hAANAT. Conditions were as described in C with the exception that the cell homogenate was incubated with p-DK14 (0.5 mM; 4°C; 30 min) before chromatography. The sequence of DK14 peptide is given in the text. The 14-3-3 and hAANAT proteins were detected as in C and p-DK14 was detected with antiserum As 2345.

Complex formation by purified p-AANAT and 14-3-3-ζ proteins. (A) Elution profile of p-AANAT (23 μg). (B) Elution profile of 14-3-3-ζ (30 μ g). (C) Elution profile of a 1:1 molar mixture of 14-3-3-ζ monomer (30 μg) and AANAT (23 μg). (D) Elution profile of a 1:1 molar mixture of 14-3-3-ζ (30 μg) monomer and p-AANAT (23 μg) (0.5:1 molar ratio of 14-3-3-ζ dimer and p-AANAT). p-AANAT coelutes with 14-3-3-ζ, representing a complex, and, in a later fraction, representing unbound p-AANAT. (E) Elution profile of a 2:1 molar mixture of 14-3-3-ζ monomer (60 μg) and p-AANAT (23 μg; 1:1 molar ratio of 14-3-3-ζ dimer and p-AANAT). p-AANAT coelutes with 14-3-3-ζ; unbound p-AANAT is not detected. (F) Role of PK-A/14-3-3 motif on elution of 14-3-3-ζ/AANAT complex. Incubation (4°C; 30 min; 50 μl) of a preformed p-AANAT/14-3-3 complex (see E) with a 25-fold excess of p-DK14 (0.5 mM) causes p-AANAT to elute as unbound protein. A portion of the p-DK14 coelutes with 14-3-3-ζ and also in later fractions as unbound peptide (a representative fraction is shown). In the same type of competition experiment, unphosphorylated DK14 neither coelutes with 14-3-3 nor displaces p-AANAT (data not shown).

14-3-3-ζ protects p-AANAT against clipping by thrombin. oAANAT or p-oAANAT (1 μg) was incubated with 0.1 units of thrombin (Roche Molecular Biochemicals) in the presence or absence of 14-3-3-ζ protein (5 μg) at 4°C for 0, 2, or 4 h (h) in a 50-μl reaction mixture containing 0.1 M Tris⋅HCl, pH 8.0. Proteins in a 5-μl volume of each sample were detected by Western blot analysis. (A) Immunodetection with an antiserum raised against oAANAT_1–205 (2819) detects a ∼18-kDa product. N-terminal sequencing of the ∼18-kDa fragment, using Edman degradation (protein sequencer model 477A, Applied Biosystems), indicated the cleavage site was at residue Arg²⁶. (B) Immunodetection with an IP antiserum raised against oAANAT_1–25 (3343). The thrombin degradation product generated in A is not detected, indicating that the N-terminal AANAT epitope has been removed.