Journal: Molecular & Cellular Proteomics : MCP
Article Title: Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ *
Figure Lengend Snippet: Effect of 14-3-3γ on TH phosphorylation and dephosphorylation. A , binding of TH phosphorylated on Ser19 by PRAK to immobilized 14-3-3γ. TH was phosphorylated to a stoichiometry of 0.17 (red) or 1.0 (blue) before preparation for injections at subunit concentrations of 5, 25, and 50 nM. Sensorgrams were scaled for illustration of kinetics. B , TH was [ 32 P]-labeled on Ser19 to different stoichiometries using PRAK before incubation with the PRAK inhibitor epigallocatechin gallate and 14-3-3γ (7.5 μM, TH-pS19:14-3-3 mixing ratio of 1:1.5). The complex was then diluted 1/10 in buffer containing high levels of shrimp alkaline phosphatase (SAP) (145 U/ml), and the temporal decay of 32 P-Ser19 was monitored as described under “Experimental Procedures.” Controls without 14-3-3 and without SAP were also measured (dotted lines). Exponential decay functions were fitted to each curve, and the corresponding rate constants were 0.089, 0.125, and 0.157 min −1 for TH-pS19 phosphorylated to 51% (○), 33% (▵), and 18.5% (□), respectively. Insignificant change in phosphorylation stoichiometry was observed in the absence of SAP (horizontal dotted lines), and a high rate of dephosphorylation was measured in the absence of 14-3-3γ (lower dotted lines). C , the phosphorylation of TH-pS19 (2.5 μM, pre-phosphorylated on Ser19 by PRAK) by PKA in the absence (○) or presence (●) of 14-3-3γ (10 μM).
Article Snippet: TH (5 μM) was then preincubated for 10 min on ice with or without 14-3-3γ (7.5 μM) prior to 5-fold dilution in dephosphorylation assay using shrimp alkaline phosphatase (SAP) (0.14 U/μl, Thermo Fisher Scientific), 25 mM Hepes, pH 7.2, 130 mM KCl, 2 mM MgCl2 , 1 mM DTT, at 15 °C or 25 °C.
Techniques: De-Phosphorylation Assay, Binding Assay, Labeling, Incubation