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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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Native MS of TH-pS19 in complex with 14-3-3γ. Native MS experiments were performed on purified TH-pS19 and 14-3-3γ and on their complexes. A, overlay of native mass spectra of the 14-3-3γ dimer (blue trace), the TH-pS19 tetramer (orange trace), and the (14-3-3γ)2:(TH)4:(14-3-3γ)2 complex (green trace) formed upon mixing of TH-pS19 tetramer with 14-3-3γ at a subunit mixing ratio of 1:3. B, native mass spectrum of the TH-pS19 tetramer mixed with 14-3-3γ at a subunit mixing ratio of 1:1. When we reduced the amount of 14-3-3γ, both the (14-3-3γ)2:(TH)4 complex and the (14-3-3γ)2:(TH)4:(14-3-3γ)2 complex were detected together with the free 14-3-3γ dimer.
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Figure 3: Native MS of TH-pS19 in complex with 14-3-3γ. Native MS experiments were performed on purified TH-pS19 and 14-3-3γ and on their complexes. A, overlay of native mass spectra of the 14-3-3γ dimer (blue trace), the TH-pS19 tetramer (orange trace), and the (14-3-3γ)2:(TH)4:(14-3-3γ)2 complex (green trace) formed upon mixing of TH-pS19 tetramer with 14-3-3γ at a subunit mixing ratio of 1:3. B, native mass spectrum of the TH-pS19 tetramer mixed with 14-3-3γ at a subunit mixing ratio of 1:1. When we reduced the amount of 14-3-3γ, both the (14-3-3γ)2:(TH)4 complex and the (14-3-3γ)2:(TH)4:(14-3-3γ)2 complex were detected together with the free 14-3-3γ dimer.

Mentions: Mixing TH-pS19 with 14-3-3γ at a 1:3 ratio (TH-pS19:14-3-3, subunit ratios) induced the formation of a 340.8-kDa complex. This mass corresponds to the tetrameric TH-pS19 bound to two dimers of 14-3-3γ (Fig. 3A). The formation of the complex (14-3-3γ)2:(TH)4:(14-3-3γ)2 was observed also for TH-pSer19pSer40 (341.0 kDa). In contrast, no complex could be observed when nonphosphorylated TH or TH-pS40 was mixed with 14-3-3γ (supplemental Fig. S4). When the amount of 14-3-3γ was decreased, an additional charge-state distribution was detected corresponding to a (14-3-3γ)2:(TH-pS19)4 complex (282.7 kDa) (Fig. 3B), but no evidence was found of TH bound to a single subunit of 14-3-3γ. The observed masses and related stoichiometries of the investigated proteins and complexes are summarized in Table II.


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)

Native MS of TH-pS19 in complex with 14-3-3γ. Native MS experiments were performed on purified TH-pS19 and 14-3-3γ and on their complexes. A, overlay of native mass spectra of the 14-3-3γ dimer (blue trace), the TH-pS19 tetramer (orange trace), and the (14-3-3γ)2:(TH)4:(14-3-3γ)2 complex (green trace) formed upon mixing of TH-pS19 tetramer with 14-3-3γ at a subunit mixing ratio of 1:3. B, native mass spectrum of the TH-pS19 tetramer mixed with 14-3-3γ at a subunit mixing ratio of 1:1. When we reduced the amount of 14-3-3γ, both the (14-3-3γ)2:(TH)4 complex and the (14-3-3γ)2:(TH)4:(14-3-3γ)2 complex were detected together with the free 14-3-3γ dimer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Native MS of TH-pS19 in complex with 14-3-3γ. Native MS experiments were performed on purified TH-pS19 and 14-3-3γ and on their complexes. A, overlay of native mass spectra of the 14-3-3γ dimer (blue trace), the TH-pS19 tetramer (orange trace), and the (14-3-3γ)2:(TH)4:(14-3-3γ)2 complex (green trace) formed upon mixing of TH-pS19 tetramer with 14-3-3γ at a subunit mixing ratio of 1:3. B, native mass spectrum of the TH-pS19 tetramer mixed with 14-3-3γ at a subunit mixing ratio of 1:1. When we reduced the amount of 14-3-3γ, both the (14-3-3γ)2:(TH)4 complex and the (14-3-3γ)2:(TH)4:(14-3-3γ)2 complex were detected together with the free 14-3-3γ dimer.
Mentions: Mixing TH-pS19 with 14-3-3γ at a 1:3 ratio (TH-pS19:14-3-3, subunit ratios) induced the formation of a 340.8-kDa complex. This mass corresponds to the tetrameric TH-pS19 bound to two dimers of 14-3-3γ (Fig. 3A). The formation of the complex (14-3-3γ)2:(TH)4:(14-3-3γ)2 was observed also for TH-pSer19pSer40 (341.0 kDa). In contrast, no complex could be observed when nonphosphorylated TH or TH-pS40 was mixed with 14-3-3γ (supplemental Fig. S4). When the amount of 14-3-3γ was decreased, an additional charge-state distribution was detected corresponding to a (14-3-3γ)2:(TH-pS19)4 complex (282.7 kDa) (Fig. 3B), but no evidence was found of TH bound to a single subunit of 14-3-3γ. The observed masses and related stoichiometries of the investigated proteins and complexes are summarized in Table II.

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