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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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Electron microscopy analysis of TH-pS19 and the TH-pS19:14-3-3 complexes.A, two-dimensional average image of TH-pS19 representing the largest population of particles. Bar at bottom right = 100 Å. B, two-dimensional average image of a minority population of particles of TH-pS19. C, glycerol gradients in the absence (left) or presence (right) of cross-linker. The asterisk points to the fraction used for the EM analysis of the TH-pS19:14-3-3 complexes. D, two-dimensional average image of the largest population of TH-pS19:14-3-3 complexes, with one 14-3-3 dimer (signaled by the white arrow) bound to the TH-pS19. E, two-dimensional average image of the minority population of TH-pS19:14-3-3 complexes, with presumably two 14-3-3 dimers bound on opposite sides of TH-pS19.
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Figure 4: Electron microscopy analysis of TH-pS19 and the TH-pS19:14-3-3 complexes.A, two-dimensional average image of TH-pS19 representing the largest population of particles. Bar at bottom right = 100 Å. B, two-dimensional average image of a minority population of particles of TH-pS19. C, glycerol gradients in the absence (left) or presence (right) of cross-linker. The asterisk points to the fraction used for the EM analysis of the TH-pS19:14-3-3 complexes. D, two-dimensional average image of the largest population of TH-pS19:14-3-3 complexes, with one 14-3-3 dimer (signaled by the white arrow) bound to the TH-pS19. E, two-dimensional average image of the minority population of TH-pS19:14-3-3 complexes, with presumably two 14-3-3 dimers bound on opposite sides of TH-pS19.

Mentions: The TH-pS19:14-3-3γ complex was further investigated using EM. First, aliquots of purified TH-pS19 were negatively stained and observed at the electron microscope. A total of 6347 particles were selected and processed, and the classification procedure revealed a major population whose average image (Fig. 4A) showed a particle of approximately 115 Å in length and 95 Å in width, in agreement with the hydrodynamic diameter of TH (about 12 nm) obtained via dynamic light scattering (data not shown). The size of the particle, together with the presence of four stain excluding masses, pointed to tetrameric TH-pS19 (represented in supplemental Fig. S1B). Another minor population represented a particle with approximate dimensions of 85-Å length and 95-Å width (Fig. 4B), which we believe to be the orthogonal view of the major population.


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)

Electron microscopy analysis of TH-pS19 and the TH-pS19:14-3-3 complexes.A, two-dimensional average image of TH-pS19 representing the largest population of particles. Bar at bottom right = 100 Å. B, two-dimensional average image of a minority population of particles of TH-pS19. C, glycerol gradients in the absence (left) or presence (right) of cross-linker. The asterisk points to the fraction used for the EM analysis of the TH-pS19:14-3-3 complexes. D, two-dimensional average image of the largest population of TH-pS19:14-3-3 complexes, with one 14-3-3 dimer (signaled by the white arrow) bound to the TH-pS19. E, two-dimensional average image of the minority population of TH-pS19:14-3-3 complexes, with presumably two 14-3-3 dimers bound on opposite sides of TH-pS19.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Electron microscopy analysis of TH-pS19 and the TH-pS19:14-3-3 complexes.A, two-dimensional average image of TH-pS19 representing the largest population of particles. Bar at bottom right = 100 Å. B, two-dimensional average image of a minority population of particles of TH-pS19. C, glycerol gradients in the absence (left) or presence (right) of cross-linker. The asterisk points to the fraction used for the EM analysis of the TH-pS19:14-3-3 complexes. D, two-dimensional average image of the largest population of TH-pS19:14-3-3 complexes, with one 14-3-3 dimer (signaled by the white arrow) bound to the TH-pS19. E, two-dimensional average image of the minority population of TH-pS19:14-3-3 complexes, with presumably two 14-3-3 dimers bound on opposite sides of TH-pS19.
Mentions: The TH-pS19:14-3-3γ complex was further investigated using EM. First, aliquots of purified TH-pS19 were negatively stained and observed at the electron microscope. A total of 6347 particles were selected and processed, and the classification procedure revealed a major population whose average image (Fig. 4A) showed a particle of approximately 115 Å in length and 95 Å in width, in agreement with the hydrodynamic diameter of TH (about 12 nm) obtained via dynamic light scattering (data not shown). The size of the particle, together with the presence of four stain excluding masses, pointed to tetrameric TH-pS19 (represented in supplemental Fig. S1B). Another minor population represented a particle with approximate dimensions of 85-Å length and 95-Å width (Fig. 4B), which we believe to be the orthogonal view of the major population.

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