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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;

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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 [32P]-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 32P-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).
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Figure 5: 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 [32P]-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 32P-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).

Mentions: The dissociation rate of the TH-pS19:14-3-3 complexes for TH-pS19L was very similar to that observed for full stoichiometry (TH-pS19H, Table I, Fig. 5A). This similarity was also observed for the smaller population of rapid dissociating complex, which had a similar size and rate constant as found for high phosphorylation levels (82% to 91%, 0.019 s−1). To fit the association rate constant, we used the concentration of phosphorylated TH subunits, which assumes that the rate-limiting step of complex formation is concentration dependent. We then obtained a higher ka value for TH-pS19L than for TH-pS19H, leading to a lower apparent Kd value for the TH-pS19L:14-3-3γ complex (Table I). The ratios of the ka values for TH-pS19L and TH-pS19H corresponded roughly to the concentration ratio obtained when correcting for the phosphorylation stoichiometry. Similarly as noted by native MS at lower phosphorylation stoichiometries, a binomial distribution predicted only 48% of the TH tetramers as completely nonphosphorylated and unable to bind 14-3-3 at 17% Ser19-phosphorylation, whereas 39% of the tetramers would contain one phosphorylated subunit.


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)

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 [32P]-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 32P-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).
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Figure 5: 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 [32P]-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 32P-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).
Mentions: The dissociation rate of the TH-pS19:14-3-3 complexes for TH-pS19L was very similar to that observed for full stoichiometry (TH-pS19H, Table I, Fig. 5A). This similarity was also observed for the smaller population of rapid dissociating complex, which had a similar size and rate constant as found for high phosphorylation levels (82% to 91%, 0.019 s−1). To fit the association rate constant, we used the concentration of phosphorylated TH subunits, which assumes that the rate-limiting step of complex formation is concentration dependent. We then obtained a higher ka value for TH-pS19L than for TH-pS19H, leading to a lower apparent Kd value for the TH-pS19L:14-3-3γ complex (Table I). The ratios of the ka values for TH-pS19L and TH-pS19H corresponded roughly to the concentration ratio obtained when correcting for the phosphorylation stoichiometry. Similarly as noted by native MS at lower phosphorylation stoichiometries, a binomial distribution predicted only 48% of the TH tetramers as completely nonphosphorylated and unable to bind 14-3-3 at 17% Ser19-phosphorylation, whereas 39% of the tetramers would contain one phosphorylated subunit.

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;

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Related in: MedlinePlus