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Protein kinase C-dependent dephosphorylation of tyrosine hydroxylase requires the B56δ heterotrimeric form of protein phosphatase 2A.

Ahn JH, Kim Y, Kim HS, Greengard P, Nairn AC - PLoS ONE (2011)

Bottom Line: In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC.In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis.Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Ewha Womans University School of Medicine, Seoul, Korea.

ABSTRACT
Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A). A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.

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Related in: MedlinePlus

Phosphorylation of B56δ at Ser566 mediates PMA-dependent dephosphorylation of Ser40 of tyrosine hydroxylase.N27 cells expressing either vector, FLAG-B56δ wt or FLAG-B56δ S566A mutant were treated with either DMSO (-) or PMA (10 nM) for 30 min. Cells were lysed and proteins were analyzed by SDS-PAGE and immunoblotting with phospho-specific antibodies to Ser40 and Ser31 in tyrosine hydroxylase (TH), and a total tyrosine hydroxylase antibody (upper panels). The bar graph shows quantification of the immunoblot data normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001, **P<0.01, ***P<0.05 compared with vehicle-treated vector control by one-way ANOVA with Newman-Keuls multiple comparison test.
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pone-0026292-g004: Phosphorylation of B56δ at Ser566 mediates PMA-dependent dephosphorylation of Ser40 of tyrosine hydroxylase.N27 cells expressing either vector, FLAG-B56δ wt or FLAG-B56δ S566A mutant were treated with either DMSO (-) or PMA (10 nM) for 30 min. Cells were lysed and proteins were analyzed by SDS-PAGE and immunoblotting with phospho-specific antibodies to Ser40 and Ser31 in tyrosine hydroxylase (TH), and a total tyrosine hydroxylase antibody (upper panels). The bar graph shows quantification of the immunoblot data normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001, **P<0.01, ***P<0.05 compared with vehicle-treated vector control by one-way ANOVA with Newman-Keuls multiple comparison test.

Mentions: N27 cells expressing either vector control, FLAG-B56δ wt, or FLAG-B56δ S566A, were treated with PMA, then the phosphorylation level of Ser40 of tyrosine hydroxylase was analyzed by immunoblotting (Figure 4). In control cells (vector alone), upon PMA stimulation the phosphorylation on Ser40 was decreased. Expression of FLAG-B56δ wt resulted in a slightly larger effect of PMA but this was not statistically significant. In contrast, in cells expressing FLAG-B56δ S566A, there was an increased basal level of phosphorylation of Ser40 which was unaffected by PMA. Phosphorylation of Ser31, which was used as a control, was unaffected by any condition examined.


Protein kinase C-dependent dephosphorylation of tyrosine hydroxylase requires the B56δ heterotrimeric form of protein phosphatase 2A.

Ahn JH, Kim Y, Kim HS, Greengard P, Nairn AC - PLoS ONE (2011)

Phosphorylation of B56δ at Ser566 mediates PMA-dependent dephosphorylation of Ser40 of tyrosine hydroxylase.N27 cells expressing either vector, FLAG-B56δ wt or FLAG-B56δ S566A mutant were treated with either DMSO (-) or PMA (10 nM) for 30 min. Cells were lysed and proteins were analyzed by SDS-PAGE and immunoblotting with phospho-specific antibodies to Ser40 and Ser31 in tyrosine hydroxylase (TH), and a total tyrosine hydroxylase antibody (upper panels). The bar graph shows quantification of the immunoblot data normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001, **P<0.01, ***P<0.05 compared with vehicle-treated vector control by one-way ANOVA with Newman-Keuls multiple comparison test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198769&req=5

pone-0026292-g004: Phosphorylation of B56δ at Ser566 mediates PMA-dependent dephosphorylation of Ser40 of tyrosine hydroxylase.N27 cells expressing either vector, FLAG-B56δ wt or FLAG-B56δ S566A mutant were treated with either DMSO (-) or PMA (10 nM) for 30 min. Cells were lysed and proteins were analyzed by SDS-PAGE and immunoblotting with phospho-specific antibodies to Ser40 and Ser31 in tyrosine hydroxylase (TH), and a total tyrosine hydroxylase antibody (upper panels). The bar graph shows quantification of the immunoblot data normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001, **P<0.01, ***P<0.05 compared with vehicle-treated vector control by one-way ANOVA with Newman-Keuls multiple comparison test.
Mentions: N27 cells expressing either vector control, FLAG-B56δ wt, or FLAG-B56δ S566A, were treated with PMA, then the phosphorylation level of Ser40 of tyrosine hydroxylase was analyzed by immunoblotting (Figure 4). In control cells (vector alone), upon PMA stimulation the phosphorylation on Ser40 was decreased. Expression of FLAG-B56δ wt resulted in a slightly larger effect of PMA but this was not statistically significant. In contrast, in cells expressing FLAG-B56δ S566A, there was an increased basal level of phosphorylation of Ser40 which was unaffected by PMA. Phosphorylation of Ser31, which was used as a control, was unaffected by any condition examined.

Bottom Line: In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC.In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis.Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Ewha Womans University School of Medicine, Seoul, Korea.

ABSTRACT
Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A). A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.

Show MeSH
Related in: MedlinePlus