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

Mol Cell Proteomics. 2014; pii: mcp.M113.035709.

Kleppe R, Rosati S, Jorge-Finnigan A, Alvira S, Ghorbani S, Haavik J, Valpuesta JM, Heck AJ, Martinez A.

Mol Cell Proteomics. 2014; pii: mcp.M113.035709Phosphorylated 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 this end, we performed native mass spectrometry (native MS) analyses of human TH (non-phosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40) or Ser19 and 40 (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 3-4 fold. All together this is consistent with a model where the pSer19 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 by native MS, and SPR 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, but rather has a lowered 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.

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