Dissection of Binding between a Phosphorylated Tyrosine Hydroxylase Peptide and 14-3-3ζ: A Complex Story Elucidated by NMR

Abstract

Human tyrosine hydroxylase activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator 14-3-3 proteins. We investigated the binding of singly or doubly phosphorylated and thiophosphorylated peptides, comprising the first 50 amino acids of human tyrosine hydroxylase, isoform 1 (hTH1), that contain the critical interaction domain, to 14-3-3?, by (31)P NMR. Single phosphorylation at S19 generates a high affinity 14-3-3? binding epitope, whereas singly S40-phosphorylated peptide interacts with 14-3-3? one order-of-magnitude weaker than the S19-phosphorylated peptide. Analysis of the binding data revealed that the 14-3-3? dimer and the S19- and S40-doubly phosphorylated peptide interact in multiple ways, with three major complexes formed: 1), a single peptide bound to a 14-3-3? dimer via the S19 phosphate with the S40 phosphate occupying the other binding site; 2), a single peptide bound to a 14-3-3? dimer via the S19 phosphorous with the S40 free in solution; or 3), a 14-3-3? dimer with two peptides bound via the S19 phosphorous to each binding site. Our system and data provide information as to the possible mechanisms by which 14-3-3 can engage binding partners that possess two phosphorylation sites on flexible tails. Whether these will be realized in any particular interacting pair will naturally depend on the details of each system.

Quantitative mapping of microtubule-associated protein 2c (MAP2c) phosphorylation and regulatory protein 14-3-3ζ-binding sites reveals key differences between MAP2c and its homolog Tau

“…However, they should not be overinterpreted because the MAP2c-14-3-3 interaction is more complex than the simple binding model used for the data fitting (35).…”

“…14-3-3 was purified as described previously (35). Two surface-exposed cysteines were mutated for alanine (C25A and C189A).…”

“…This allowed us to work with highly concentrated samples over longer times without the risk of disulfide bond formation. We showed earlier that the mutation of these two exposed cysteines does not change the fold or stability of the protein (35). The purity of the final protein samples, including phosphorylated and labeled samples, was checked by MALDI-MS.…”

Structural insights into the regulation of aromatic amino acid hydroxylation

“…Tight binding of TyrH1 to a 14-3-3 protein requires that Ser19 be phosphorylated, and the binding involves the flexible N-terminus of the regulatory domain [34,35]. The stoichiometry of binding is consistent with one 14-3-3 dimer binding one TyrH regulatory dimer in which both Ser19s are phosphorylated.…”

A new role for α‐synuclein in Parkinson's disease: Alteration of ER–mitochondrial communication

“…Tyrosine hydroxylase, a homotetramer, 90 has two forms: an "active" form, which is phosphorylated by a variety of kinases, 91 primarily at Ser40 92,93 (and potentiated by a prior phosphorylation at Ser19 91,94 ), and an "inactive," unphosphorylated form, either synthesized de novo or generated by dephosphorylation of the active form by protein phosphatase 2A (PP2A). 91,[95][96][97][98] The phosphorylation of TH, first at Ser19 and then at Ser40, is mediated by its binding to 14-3-3, 99,100 one of seven related proteins (denoted b/ a, g, e, h, f, r, and s/u) 101 ; the exact isoform(s) that bind TH is a matter of some debate, with data in support of g 102,103 and f, [104][105][106][107] although we note that 14-3-3f is highly abundant in ventral midbrain 107 (containing the SN) and is the predominant isoform present in the caudate-putamen as determined by immunocytochemistry of mouse brain. 108 Like TH, a-Syn also exists in two forms based on whether it is, or is not, phosphorylated, mainly at Ser129.…”