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

• Published in: The Journal of biological chemistry Vol. 284; no. 47; pp. 32758 - 32769
• Main Authors: Halskau, Øyvind, Ying, Ming, Baumann, Anne, Kleppe, Rune, Rodriguez-Larrea, David, Almås, Bjørg, Haavik, Jan, Martinez, Aurora
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.11.2009; American Society for Biochemistry and Molecular Biology
• Subjects: 14-3-3 Proteins - chemistry; Amino Acid Sequence; Cell Membrane - metabolism; Chromaffin Cells - cytology; Humans; Hydrogen-Ion Concentration; Kinetics; Levodopa - chemistry; Lipids and Lipoproteins: Metabolism, Regulation, and Signaling; Molecular Sequence Data; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Surface Plasmon Resonance; Tyrosine 3-Monooxygenase - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.027706

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1–43)) and Ser19-phosphorylated (THp-(1–43)) states by surface plasmon resonance. THp-(1–43) showed high affinity for 14-3-3 proteins (Kd ∼ 0.5 μm for 14-3-3γ and -ζ and 7 μm for 14-3-3η). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S0.5 = 25–58 μm (TH-(1–43)) and S0.5 = 135–475 μm (THp-(1–43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3γ showed a preferential binding to membranes, compared with 14-3-3ζ, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3γ for negatively charged membranes (S0.5 = 1–9 μm) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser19-phosphorylated TH, 14-3-3γ, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of l-DOPA and dopamine synthesis.