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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 PAGE and immunodetection of 14-3-3γ, TH, and phosphorylated forms of TH, alone and in complex. TH (TH, TH-pS19, or TH-pS19pS40) and 14-3-3γ complexes, formed by incubation of TH:14-3-3 at a mixing ratio of 1:3, were separated via native PAGE and transferred to nitrocellulose membranes. Ponceau Red staining was used to visualize all protein forms, and specific antibodies anti-TH and anti-14-3-3γ were used for TH and 14-3-3γ detection.
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Figure 2: Native PAGE and immunodetection of 14-3-3γ, TH, and phosphorylated forms of TH, alone and in complex. TH (TH, TH-pS19, or TH-pS19pS40) and 14-3-3γ complexes, formed by incubation of TH:14-3-3 at a mixing ratio of 1:3, were separated via native PAGE and transferred to nitrocellulose membranes. Ponceau Red staining was used to visualize all protein forms, and specific antibodies anti-TH and anti-14-3-3γ were used for TH and 14-3-3γ detection.

Mentions: In order to further investigate complex formation between the different forms of TH and 14-3-3γ, we performed native PAGE. Native electrophoresis using gradient native gels and BisTris and Tricine running buffers gave the best resolution, although band positions in the gel could not be assigned to precise molecular weights. The proteins were transferred from the gels to nitrocellulose membranes and stained with Ponceau Red before specific immunodetection of TH or 14-3-3γ. TH, as well as its two phosphorylated forms, showed an intense upper band and a lower, more diffuse band (Fig. 2) that could be associated with tetrameric TH and a minor dimeric form of the enzyme, respectively. The acidic 14-3-3γ ran closer to the front of the gel and displayed a double band pattern. The incubation of TH-pS19 or TH-pS19pS40 with 14-3-3γ at a TH:14-3-3γ subunit ratio of 1:3 led to a similar upward shift of both bands of TH, and the lower band became sharper and more intense. The incubation of nonphosphorylated TH with 14-3-3γ did not render any band differences indicating that complex formation requires TH phosphorylation.


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 PAGE and immunodetection of 14-3-3γ, TH, and phosphorylated forms of TH, alone and in complex. TH (TH, TH-pS19, or TH-pS19pS40) and 14-3-3γ complexes, formed by incubation of TH:14-3-3 at a mixing ratio of 1:3, were separated via native PAGE and transferred to nitrocellulose membranes. Ponceau Red staining was used to visualize all protein forms, and specific antibodies anti-TH and anti-14-3-3γ were used for TH and 14-3-3γ detection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Native PAGE and immunodetection of 14-3-3γ, TH, and phosphorylated forms of TH, alone and in complex. TH (TH, TH-pS19, or TH-pS19pS40) and 14-3-3γ complexes, formed by incubation of TH:14-3-3 at a mixing ratio of 1:3, were separated via native PAGE and transferred to nitrocellulose membranes. Ponceau Red staining was used to visualize all protein forms, and specific antibodies anti-TH and anti-14-3-3γ were used for TH and 14-3-3γ detection.
Mentions: In order to further investigate complex formation between the different forms of TH and 14-3-3γ, we performed native PAGE. Native electrophoresis using gradient native gels and BisTris and Tricine running buffers gave the best resolution, although band positions in the gel could not be assigned to precise molecular weights. The proteins were transferred from the gels to nitrocellulose membranes and stained with Ponceau Red before specific immunodetection of TH or 14-3-3γ. TH, as well as its two phosphorylated forms, showed an intense upper band and a lower, more diffuse band (Fig. 2) that could be associated with tetrameric TH and a minor dimeric form of the enzyme, respectively. The acidic 14-3-3γ ran closer to the front of the gel and displayed a double band pattern. The incubation of TH-pS19 or TH-pS19pS40 with 14-3-3γ at a TH:14-3-3γ subunit ratio of 1:3 led to a similar upward shift of both bands of TH, and the lower band became sharper and more intense. The incubation of nonphosphorylated TH with 14-3-3γ did not render any band differences indicating that complex formation requires TH phosphorylation.

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