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 [32P]-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.
Highlights
From the ‡Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway; §K
Tyrosine hydroxylase (TH) Purification and Phosphorylation—The TH tetramer is structurally organized as a dimer of dimers, an organization that may allow for a number of possible complexes between phosphorylated TH and 14-3-3
We have previously described the phosphorylation of TH by PRAK, which has the highest selectivity for Ser19 among the currently known TH kinases [20]
Summary
Protein Expression and Purification—The construct (His)6-ZZ-hTH1 was prepared by cloning the human TH1 gene in the pET-ZZ-1a vector [44].2 The construct, which codes for a fusion protein, with a Tobacco etch virus protease-cutting site between the N-terminal His-ZZ fusion partner and hTH1, was expressed in Escherichia coli (BL21 Codon Plus(DE3), Stratagene, La Jolla, CA) in auto-induction media at 37 °C overnight [45]. Phosphorylation of TH for dephosphorylation studies was performed at the following conditions: TH (2 mg/ml), PRAK (7 U/ml), 25 mM Hepes, pH 7.2, 130 mM KCl, 0.1 mM EGTA, 0.1 mM ATP, 0.01 mCi [␥-32P]ATP (PerkinElmer), 5 mM MgCl2, 10% glycerol, 1 mM DTT, 25 °C. For measurement of the rate of phosphorylation of TH in the presence or absence of 14-3-3␥ by PRAK or PKA, we used the following conditions: TH (2.5 M), PRAK (5 U/ml), PKA (1 nM catalytic subunit), 14-3-3␥ (10 M), 25 mM Hepes, pH 7.2, 130 mM KCl, 0.1 mM EGTA, 0.1 mM ATP, 0.01 mCi [␥-32P]ATP (PerkinElmer), 5 mM MgCl2, 10% glycerol, 1 mM DTT, 0.5 mg/ml BSA, 0.1 mg/ml soya bean trypsin inhibitor, 25 °C. The dissociation rate constant, representing the highest population of complexes (Ͼ80%), was used to fit the association rate constants
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