Kleppe Rune, Rosati Sara, Jorge-Finnigan Ana, Alvira Sara, Ghorbani Sadaf, Haavik Jan, Valpuesta José María, Heck Albert J R, Martinez Aurora
From the ‡Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway; §K. G. Jebsen Centre for Research on Neuropsychiatric disorders, Jonas Lies vei 91, 5009 Bergen, Norway; ¶Division for Psychiatry, Haukeland University Hospital, Sandviksleitet 1, 5036 Bergen, Norway;
**Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; ‡‡Netherland Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands;
Mol Cell Proteomics. 2014 Aug;13(8):2017-30. doi: 10.1074/mcp.M113.035709. Epub 2014 Jun 19.
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.
磷酸化酪氨酸羟化酶（TH）可与14-3-3蛋白形成复合物，从而导致酶的激活和稳定。尽管TH是最早被鉴定为这些普遍存在的调节蛋白的结合伙伴之一，但其结合化学计量和激活机制仍不清楚。为了解决这个问题，我们对人TH（未磷酸化或在Ser19（TH-pS19）、Ser40（TH-pS40）或Ser19和Ser40（TH-pS19pS40）处磷酸化）单独以及与14-3-3γ一起进行了天然质谱分析。四聚体TH-pS19（224 kDa）与14-3-3γ（58.3 kDa）以高亲和力（Kd = 3.2 nM）结合，形成含有一个（282.4 kDa）或两个（340.8 kDa）14-3-3二聚体的复合物。电子显微镜还揭示了一个主要的不对称复合物群体，与一个TH四聚体和一个14-3-3二聚体一致，以及一个次要的由一个TH四聚体和两个14-3-3二聚体组成的对称复合物群体。较低的磷酸化化学计量（0.15 - 0.54磷酸/单体）在结合动力学上产生了适度变化，但天然质谱检测到的对称TH:14-3-3γ复合物要少得多。有趣的是对于低磷酸化化学计量（0.18 - 0.52），[(32)P]-TH-pS19的去磷酸化是单指数的，并且添加磷酸酶使TH-pS19:14-3-3γ复合物的解离加速了3至4倍。所有这些都与一个模型一致，即TH四聚体中的pS19残基在与14-3-3γ的结合中贡献不同。通过天然质谱未检测到TH-pS40与14-3-3γ之间的复合物形成，表面等离子体共振显示这种相互作用非常弱。此外，TH-pS19pS40在结合化学计量和亲和力方面（Kd = 2.1 nM）与TH-pS19表现相似。然而，我们发现14-3-3γ抑制了PKA对TH-pS19在Ser40处的磷酸化速率（3.5倍）。因此我们得出结论，Ser40对14-3-3γ的结合没有显著贡献，而是在TH:14-3-3γ复合物中的可及性降低。这增加了我们对TH精细调节的生理调节的理解，包括多个位点的分级磷酸化。
