Phosphorylated nitrate reductase and 14-3-3 proteins. Site of interaction, effects of ions, and evidence for an amp-binding site on 14-3-3 proteins

Abstract

The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14omega, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14omega, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5' isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5'-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14omega.

Figure 1

Disruption of the pNR:14-3-3 inactive complex by three phosphorylated synthetic peptides, pNR6, pNR24, and pNR25. Partially purified NR was phosphorylated and immediately desalted, as described in Methods. The mixtures of pNR, GF14ω (5 μm), and increasing concentrations of the indicated phosphopeptide were preincubated at 25°C for 5 min before assaying for NRA. Activities are expressed as a percentage of the control, which contained no phosphopeptides or GF14ω, pH 7.5, plus 5 mm Mg²⁺. Values are means of three determinants ± se. See Table I for peptide sequences.

Figure 2

The physical effectors Pi and KCl inhibit the binding of pNR6 to GF14ω at pH 6.5. GF14ω (500 pmol) was incubated with 60 pmol of [³²P]pNR6 (60 cpm/pmol) plus additions as shown. Representative results are shown.

Figure 3

Ionic strength affects the surface hydrophobicity of 14-3-3 proteins. Emission spectra of bis-ANS, a fluorescent probe, to monitor changes in GF14ω surface hydrophobicity. A final concentration of 5 μm GF14ω in 100 mm Mops, pH 7.5, 10 mm Mg²⁺, with the addition of 1 μm bis-ANS, was used as the control. To this mixture 50 or 100 mm KCl was added, and the bis-ANS emission spectrum was recorded. The excitation wavelength was 385 nm, and the emission spectra were recorded from 400 to 600 nm.

Figure 4

Concentration-dependent disruption of the pNR6:14-3-3 association by 5′-AMP in the presence or absence of 5 mm Mg²⁺. Either a spinach 14-3-3 protein mixture (1100 pmol) or recombinant GF14ω (900 pmol) was incubated with [³²P]pNR6 (100 pmol; 100 cpm/pmol), as described in Methods. Results are expressed as a percentage of the maximum binding in the controls, which were 58 pmol (A), 18 pmol (B), 28 pmol (C), and 13 pmol (D), respectively.

Figure 5

The fluorescence emission spectra of TNP-AMP in the presence of GF14ω, aldolase, and carbonic anhydrase. A, Intrinsic fluorescence of 40 μm TNP-AMP in 100 mm Mops, pH 7.5, 10 mm Mg²⁺ (Control), followed by the addition of 150 μg of GF14ω. The inset shows the titration of 0 to 40 μm TNP-AMP in the presence or absence of 10 mm Mg²⁺. B, Intrinsic fluorescence of TNP-AMP in the absence of protein (Control), and two spectra after the addition of 150 μg of aldolase or carbonic anhydrase. For all assays, the excitation wavelength was 410 nm and the emission spectra was recorded from 450 to 600 nm, with maximal intensity at approximately 543 nm. Before recording the emission spectra a preincubation of 5 min at 22°C after the addition of TNP-AMP was allowed.