title: 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.
authors.affiliation: 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;
authors.forename: Rune
authors.initials: R
authors.lastname: Kleppe
authors.affiliation: **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;
authors.forename: Sara
authors.initials: S
authors.lastname: Rosati
authors.affiliation: From the ‡Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway;
authors.forename: Ana
authors.initials: A
authors.lastname: Jorge-Finnigan
authors.affiliation: §§Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049 Madrid, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Alvira
authors.affiliation: From the ‡Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway;
authors.forename: Sadaf
authors.initials: S
authors.lastname: Ghorbani
authors.affiliation: 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;
authors.forename: Jan
authors.initials: J
authors.lastname: Haavik
authors.affiliation: §§Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049 Madrid, Spain.
authors.forename: José María
authors.initials: JM
authors.lastname: Valpuesta
authors.affiliation: **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; aurora.martinez@biomed.uib.no A.J.R.Heck@uu.nl.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.affiliation: 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; aurora.martinez@biomed.uib.no A.J.R.Heck@uu.nl.
authors.forename: Aurora
authors.initials: A
authors.lastname: Martinez
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D010766:Phosphorylation; D055503:Protein Multimerization; D012694:Serine / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D012694:Serine; D014446:Tyrosine 3-Monooxygenase