Phosphorylation dependence and stoichiometry of the complex formed by tyrosine hydroxylase and 14-3-3γ

Abstract

Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15-0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [(32)P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18-0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in the TH:14-3-3γ complex. This adds to our understanding of the fine-tuned physiological regulation of TH, including hierarchical phosphorylation at multiple sites.

Fig. 1.

Binding of different phosphorylated forms of TH to 14-3-3γ by SPR. 14-3-3γ was immobilized via amine coupling as described under “Experimental Procedures.” TH-pS19 (A) and TH-pS19pS40 (B) were injected at different concentrations (10 nM (black, a), 25 nM (red, b), 50 nM (blue, c), and 100 nM (green, d)), and the association to 14-3-3γ was monitored in terms of response units. C, GST-14-3-3γ was immobilized to a CM5 chip (GE Healthcare) using a GST-immobilization kit (GE Healthcare) according to recommendations by the manufacturer. Sensorgrams were recorded at a flow rate of 30 μl/min, and the figure compares injected (90 μl) nonphosphorylated TH (100 nM, black), TH-pS40 (100 nM, blue), and TH-pS19 (100 nM, red).

Fig. 2.

Native PAGE and immunodetection of 14-3-3γ, TH, and phosphorylated forms of TH, alone and in complex. TH (TH, TH-pS19, or TH-pS19pS40) and 14-3-3γ complexes, formed by incubation of TH:14-3-3 at a mixing ratio of 1:3, were separated via native PAGE and transferred to nitrocellulose membranes. Ponceau Red staining was used to visualize all protein forms, and specific antibodies anti-TH and anti-14-3-3γ were used for TH and 14-3-3γ detection.

Fig. 3.

Native MS of TH-pS19 in complex with 14-3-3γ. Native MS experiments were performed on purified TH-pS19 and 14-3-3γ and on their complexes. A, overlay of native mass spectra of the 14-3-3γ dimer (blue trace), the TH-pS19 tetramer (orange trace), and the (14-3-3γ)₂:(TH)₄:(14-3-3γ)₂ complex (green trace) formed upon mixing of TH-pS19 tetramer with 14-3-3γ at a subunit mixing ratio of 1:3. B, native mass spectrum of the TH-pS19 tetramer mixed with 14-3-3γ at a subunit mixing ratio of 1:1. When we reduced the amount of 14-3-3γ, both the (14-3-3γ)₂:(TH)₄ complex and the (14-3-3γ)₂:(TH)₄:(14-3-3γ)₂ complex were detected together with the free 14-3-3γ dimer.

Fig. 4.

Electron microscopy analysis of TH-pS19 and the TH-pS19:14-3-3 complexes. A, two-dimensional average image of TH-pS19 representing the largest population of particles. Bar at bottom right = 100 Å. B, two-dimensional average image of a minority population of particles of TH-pS19. C, glycerol gradients in the absence (left) or presence (right) of cross-linker. The asterisk points to the fraction used for the EM analysis of the TH-pS19:14-3-3 complexes. D, two-dimensional average image of the largest population of TH-pS19:14-3-3 complexes, with one 14-3-3 dimer (signaled by the white arrow) bound to the TH-pS19. E, two-dimensional average image of the minority population of TH-pS19:14-3-3 complexes, with presumably two 14-3-3 dimers bound on opposite sides of TH-pS19.

Fig. 5.

Effect of 14-3-3γ on TH phosphorylation and dephosphorylation. A, binding of TH phosphorylated on Ser19 by PRAK to immobilized 14-3-3γ. TH was phosphorylated to a stoichiometry of 0.17 (red) or 1.0 (blue) before preparation for injections at subunit concentrations of 5, 25, and 50 nM. Sensorgrams were scaled for illustration of kinetics. B, TH was [³²P]-labeled on Ser19 to different stoichiometries using PRAK before incubation with the PRAK inhibitor epigallocatechin gallate and 14-3-3γ (7.5 μM, TH-pS19:14-3-3 mixing ratio of 1:1.5). The complex was then diluted 1/10 in buffer containing high levels of shrimp alkaline phosphatase (SAP) (145 U/ml), and the temporal decay of ³²P-Ser19 was monitored as described under “Experimental Procedures.” Controls without 14-3-3 and without SAP were also measured (dotted lines). Exponential decay functions were fitted to each curve, and the corresponding rate constants were 0.089, 0.125, and 0.157 min⁻¹ for TH-pS19 phosphorylated to 51% (○), 33% (▵), and 18.5% (□), respectively. Insignificant change in phosphorylation stoichiometry was observed in the absence of SAP (horizontal dotted lines), and a high rate of dephosphorylation was measured in the absence of 14-3-3γ (lower dotted lines). C, the phosphorylation of TH-pS19 (2.5 μM, pre-phosphorylated on Ser19 by PRAK) by PKA in the absence (○) or presence (●) of 14-3-3γ (10 μM).