Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

Halskau, Øyvind; Ying, Ming; Baumann, Anne; Kleppe, Rune; Rodríguez-Larrea, David; Almås, Bjørg; Haavik, Jan; Martı́nez, Aurora

doi:10.1074/jbc.m109.027706

Cited by 41 publications

(82 citation statements)

References 75 publications

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“…Bacteria were lysed by French press, and GST–14-3-3 fusion proteins were purified on glutathione Sepharose 4B (GE Healthcare) as described previously (30). …”

Section: Methodsmentioning

confidence: 99%

Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ

Kleppe

Rosati

Jorge‐Finnigan

et al. 2014

Molecular & Cellular Proteomics

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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.

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“…Bacteria were lysed by French press, and GST–14-3-3 fusion proteins were purified on glutathione Sepharose 4B (GE Healthcare) as described previously (30). …”

Section: Methodsmentioning

confidence: 99%

Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ

Kleppe

Rosati

Jorge‐Finnigan

et al. 2014

Molecular & Cellular Proteomics

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“…TH is a cytoplasmic protein but it was also found to be membrane-bound at the brain nerve endings and synaptic vesicles1819. There is scant knowledge on the structural determinants and conformational changes occurring upon TH membrane binding, though it has been shown that the direct interaction with negatively charged membranes depends on N-terminal extension to the ACT domain2021.…”

mentioning

confidence: 99%

“…Based on the propensity of TH to bind to negatively charged membranes2021 in this work we have studied the effects that this interaction may have on membrane integrity, enzyme conformation and cellular viability. We have examined the interaction of isoform 1 of human TH (hTH1) with negatively charged liposomes using large unilamellar vesicles (LUVs).…”

mentioning

confidence: 99%

Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity

Baumann

Jorge‐Finnigan

Jung‐KC

et al. 2016

Sci Rep

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Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes. Binding to both large and giant unilamellar vesicles causes membrane permeabilization, as observed by efflux and influx of fluorescence dyes. Whereas the initial protein-membrane interaction involves the N-terminal tail that constitutes an extension of the regulatory ACT-domain, prolonged membrane binding induces misfolding and self-oligomerization of TH over time as shown by circular dichroism and Thioflavin T fluorescence. The gradual amyloid-like aggregation likely occurs through cross-β interactions involving aggregation-prone motives in the catalytic domains, consistent with the formation of chain and ring-like protofilaments observed by atomic force microscopy in monolayer-bound TH. PC12 cells treated with the neurotoxin 6-hydroxydopamine displayed increased TH levels in the mitochondrial fraction, while incubation of isolated mitochondria with TH led to a decrease in the mitochondrial membrane potential. Furthermore, cell-substrate impedance and viability assays showed that supplementing the culture media with TH compromises cell viability over time. Our results revealed that the disruptive effect of TH on cell membranes may be a cytotoxic and pathogenic factor if the regulation and intracellular stability of TH is compromised.

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“…This mix is considered a good mimic of negatively charged physiologically relevant membranes. 30,33 Deposition of mixed polypeptide-lipid monolayer films on hydrophilic mica results in an orientation of the lipid head groups toward the mica surface. 34 Thus, the interacting polypeptides are expected to be trapped between the mica and the lipid monolayers or, alternatively, intercalated in the lipid film.…”

Section: Membrane Distortions and Formation Of Aos By Hamlet Studied mentioning

confidence: 99%

HAMLET Forms Annular Oligomers When Deposited with Phospholipid Monolayers

Baumann

Gjerde

Ying

et al. 2012

Journal of Molecular Biology

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Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH-as observed by liposome content leakage and circular dichroism experiments-and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.

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Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

Cited by 41 publications

References 75 publications

Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ

Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ

Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity

HAMLET Forms Annular Oligomers When Deposited with Phospholipid Monolayers

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