Three-way interaction between 14-3-3 proteins, the N-terminal region of tyrosine hydroxylase, and negatively charged membranes.
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 Ser(19)-phosphorylated (THp-(1-43)) states by surface plasmon resonance. THp-(1-43) showed high affinity for 14-3-3 proteins (K(d) approximately 0.5 microM for 14-3-3gamma and -zeta and 7 microM for 14-3-3eta). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S(0.5) = 25-58 microM (TH-(1-43)) and S(0.5) = 135-475 microM (THp-(1-43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3gamma showed a preferential binding to membranes, compared with 14-3-3zeta, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3gamma for negatively charged membranes (S(0.5) = 1-9 microM) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser(19)-phosphorylated TH, 14-3-3gamma, 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.
Department of Biomedicine, University of Bergen, Jonas Lies Vei 91, N-5009 Bergen, Norway.
Øyvind
Ø
Halskau
Ming
M
Ying
Anne
A
Baumann
Rune
R
Kleppe
David
D
Rodriguez-Larrea
Bjørg
B
Almås
Jan
J
Haavik
Aurora
A
Martinez
D048948:14-3-3 Proteins / Q000737:chemistry*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D019439:Chromaffin Cells / Q000166:cytology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007980:Levodopa / Q000737:chemistry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry*
D048948:14-3-3 Proteins; D007980:Levodopa; D014446:Tyrosine 3-Monooxygenase