Limits...
Phosphorylation dependence and stoichiometry of the complex formed by tyrosine hydroxylase and 14-3-3γ.

Kleppe R, Rosati S, Jorge-Finnigan A, Alvira S, Ghorbani S, Haavik J, Valpuesta JM, Heck AJ, Martinez A - Mol. Cell Proteomics (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway; §K. G. Jebsen Centre for Research on Neuropsychiatric disorders, Jonas Lies vei 91, 5009 Bergen, Norway; ¶Division for Psychiatry, Haukeland University Hospital, Sandviksleitet 1, 5036 Bergen, Norway;

Show MeSH

Related in: MedlinePlus

Binding of different phosphorylated forms of TH to 14-3-3γ by SPR. 14-3-3γ was immobilized via amine coupling as described under “Experimental Procedures.” TH-pS19 (A) and TH-pS19pS40 (B) were injected at different concentrations (10 nM (black, a), 25 nM (red, b), 50 nM (blue, c), and 100 nM (green, d)), and the association to 14-3-3γ was monitored in terms of response units. C, GST-14-3-3γ was immobilized to a CM5 chip (GE Healthcare) using a GST-immobilization kit (GE Healthcare) according to recommendations by the manufacturer. Sensorgrams were recorded at a flow rate of 30 μl/min, and the figure compares injected (90 μl) nonphosphorylated TH (100 nM, black), TH-pS40 (100 nM, blue), and TH-pS19 (100 nM, red).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4125734&req=5

Figure 1: Binding of different phosphorylated forms of TH to 14-3-3γ by SPR. 14-3-3γ was immobilized via amine coupling as described under “Experimental Procedures.” TH-pS19 (A) and TH-pS19pS40 (B) were injected at different concentrations (10 nM (black, a), 25 nM (red, b), 50 nM (blue, c), and 100 nM (green, d)), and the association to 14-3-3γ was monitored in terms of response units. C, GST-14-3-3γ was immobilized to a CM5 chip (GE Healthcare) using a GST-immobilization kit (GE Healthcare) according to recommendations by the manufacturer. Sensorgrams were recorded at a flow rate of 30 μl/min, and the figure compares injected (90 μl) nonphosphorylated TH (100 nM, black), TH-pS40 (100 nM, blue), and TH-pS19 (100 nM, red).

Mentions: Using SPR, we measured the binding affinity of TH-pS19 and TH-pS19pS40 to 14-3-3γ immobilized by amine coupling (Figs. 1A and 1B). We did not observe any binding to 14-3-3γ when we used nonphosphorylated TH, and we noted very low binding with TH-pS40 (Fig. 1C). Injections of different concentrations of TH (1–100 nM) were performed for at least two different immobilizations, and the resulting sensorgrams were analyzed to obtain the dissociation and association rate constants (Table I). As described previously, we consistently observed heterogeneity in the dissociation rate constant, possibly arising from two different populations, where the larger population (83% to 92%) had a slow dissociation rate constant (Table I) and the remainder had a rate constant of 0.018 ± 0.004 s−1. Using the dissociation rate constant from the larger population, we estimated association rate constants (ka) and the resulting equilibrium dissociation constant (Kd) for the protein interactions (Table I). Thus, we estimated a Kd value of ∼3.2 nM or ∼2.1 nM for the interaction between 14-3-3γ and TH-pS19 or TH-pS19pS40, respectively. The rate constants were also very similar for the two types of complexes, suggesting that these phospho-species of TH associate with similar strength and kinetics to 14-3-3γ. It therefore seemed unlikely that 14-3-3γ engaged in different modes of binding for TH phosphorylated on only Ser19 than for TH with both Ser19 and Ser40 phosphorylated (Table I). The phospho-Ser recognition configurations noted in parts iii and iv of supplemental Fig. S1C thus appear unlikely.


Phosphorylation dependence and stoichiometry of the complex formed by tyrosine hydroxylase and 14-3-3γ.

Kleppe R, Rosati S, Jorge-Finnigan A, Alvira S, Ghorbani S, Haavik J, Valpuesta JM, Heck AJ, Martinez A - Mol. Cell Proteomics (2014)

Binding of different phosphorylated forms of TH to 14-3-3γ by SPR. 14-3-3γ was immobilized via amine coupling as described under “Experimental Procedures.” TH-pS19 (A) and TH-pS19pS40 (B) were injected at different concentrations (10 nM (black, a), 25 nM (red, b), 50 nM (blue, c), and 100 nM (green, d)), and the association to 14-3-3γ was monitored in terms of response units. C, GST-14-3-3γ was immobilized to a CM5 chip (GE Healthcare) using a GST-immobilization kit (GE Healthcare) according to recommendations by the manufacturer. Sensorgrams were recorded at a flow rate of 30 μl/min, and the figure compares injected (90 μl) nonphosphorylated TH (100 nM, black), TH-pS40 (100 nM, blue), and TH-pS19 (100 nM, red).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125734&req=5

Figure 1: Binding of different phosphorylated forms of TH to 14-3-3γ by SPR. 14-3-3γ was immobilized via amine coupling as described under “Experimental Procedures.” TH-pS19 (A) and TH-pS19pS40 (B) were injected at different concentrations (10 nM (black, a), 25 nM (red, b), 50 nM (blue, c), and 100 nM (green, d)), and the association to 14-3-3γ was monitored in terms of response units. C, GST-14-3-3γ was immobilized to a CM5 chip (GE Healthcare) using a GST-immobilization kit (GE Healthcare) according to recommendations by the manufacturer. Sensorgrams were recorded at a flow rate of 30 μl/min, and the figure compares injected (90 μl) nonphosphorylated TH (100 nM, black), TH-pS40 (100 nM, blue), and TH-pS19 (100 nM, red).
Mentions: Using SPR, we measured the binding affinity of TH-pS19 and TH-pS19pS40 to 14-3-3γ immobilized by amine coupling (Figs. 1A and 1B). We did not observe any binding to 14-3-3γ when we used nonphosphorylated TH, and we noted very low binding with TH-pS40 (Fig. 1C). Injections of different concentrations of TH (1–100 nM) were performed for at least two different immobilizations, and the resulting sensorgrams were analyzed to obtain the dissociation and association rate constants (Table I). As described previously, we consistently observed heterogeneity in the dissociation rate constant, possibly arising from two different populations, where the larger population (83% to 92%) had a slow dissociation rate constant (Table I) and the remainder had a rate constant of 0.018 ± 0.004 s−1. Using the dissociation rate constant from the larger population, we estimated association rate constants (ka) and the resulting equilibrium dissociation constant (Kd) for the protein interactions (Table I). Thus, we estimated a Kd value of ∼3.2 nM or ∼2.1 nM for the interaction between 14-3-3γ and TH-pS19 or TH-pS19pS40, respectively. The rate constants were also very similar for the two types of complexes, suggesting that these phospho-species of TH associate with similar strength and kinetics to 14-3-3γ. It therefore seemed unlikely that 14-3-3γ engaged in different modes of binding for TH phosphorylated on only Ser19 than for TH with both Ser19 and Ser40 phosphorylated (Table I). The phospho-Ser recognition configurations noted in parts iii and iv of supplemental Fig. S1C thus appear unlikely.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway; §K. G. Jebsen Centre for Research on Neuropsychiatric disorders, Jonas Lies vei 91, 5009 Bergen, Norway; ¶Division for Psychiatry, Haukeland University Hospital, Sandviksleitet 1, 5036 Bergen, Norway;

Show MeSH
Related in: MedlinePlus