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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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PMA-dependent dephosphorylation of Ser40 in tyrosine hyroxylase is inhibited by down-regulation of B56δ.N27 cells were infected with either AAV-control RNAi or AAV-B56δ RNAi for 48 h. N27 cells were then pretreated with either vehicle or rottlerin (5 µM) for 30 min, then treated with PMA (10 nM) for 10 min as indicated. Phosphorylation of Ser40 and Ser31 of tyrosine hyroxylase was measured as above. B56δ levels were measured by immunoblotting. The bar graph shows quantification of immunoblot data normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001 compared with vehicle-treated control by one-way ANOVA with Newman-Keuls multiple comparison test.
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pone-0026292-g005: PMA-dependent dephosphorylation of Ser40 in tyrosine hyroxylase is inhibited by down-regulation of B56δ.N27 cells were infected with either AAV-control RNAi or AAV-B56δ RNAi for 48 h. N27 cells were then pretreated with either vehicle or rottlerin (5 µM) for 30 min, then treated with PMA (10 nM) for 10 min as indicated. Phosphorylation of Ser40 and Ser31 of tyrosine hyroxylase was measured as above. B56δ levels were measured by immunoblotting. The bar graph shows quantification of immunoblot data normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001 compared with vehicle-treated control by one-way ANOVA with Newman-Keuls multiple comparison test.

Mentions: These results suggest that the FLAG-B56δ S566A mutant may be acting in a dominant-negative manner to antagonize the endogenous action of B56δ. We further examined the PKC-dependent regulation of PP2A/B56δ using RNAi to down-regulate endogenous B56δ expression in N27 cells (Figure 5). B56δ-specific RNAi, expressed via adeno-associated virus (AAV) in N27 cells, resulted in reduced expression of endogenous B56δ protein by the B56δ-specific RNAi (less than 20% compared to control RNAi). Control RNAi expression had no effect compared to non-infected cells (not shown). Control or B56δ knock-down N27 cells were treated with DMSO vehicle, PMA or PMA together with rottlerin. PMA treatment resulted in dephosphorylation of phospho-Ser40 in control RNAi-infected cells, and this was inhibited by rottlerin. However, there was no effect of PMA treatment in cells in which B56δ expression was knocked down. Notably, the basal level of phospho-Ser40 was increased following knockdown of B56δ. Phosphorylation of Ser31 was unaffected under any of the conditions used.


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)

PMA-dependent dephosphorylation of Ser40 in tyrosine hyroxylase is inhibited by down-regulation of B56δ.N27 cells were infected with either AAV-control RNAi or AAV-B56δ RNAi for 48 h. N27 cells were then pretreated with either vehicle or rottlerin (5 µM) for 30 min, then treated with PMA (10 nM) for 10 min as indicated. Phosphorylation of Ser40 and Ser31 of tyrosine hyroxylase was measured as above. B56δ levels were measured by immunoblotting. The bar graph shows quantification of immunoblot data normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001 compared with vehicle-treated control by one-way ANOVA with Newman-Keuls multiple comparison test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198769&req=5

pone-0026292-g005: PMA-dependent dephosphorylation of Ser40 in tyrosine hyroxylase is inhibited by down-regulation of B56δ.N27 cells were infected with either AAV-control RNAi or AAV-B56δ RNAi for 48 h. N27 cells were then pretreated with either vehicle or rottlerin (5 µM) for 30 min, then treated with PMA (10 nM) for 10 min as indicated. Phosphorylation of Ser40 and Ser31 of tyrosine hyroxylase was measured as above. B56δ levels were measured by immunoblotting. The bar graph shows quantification of immunoblot data normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001 compared with vehicle-treated control by one-way ANOVA with Newman-Keuls multiple comparison test.
Mentions: These results suggest that the FLAG-B56δ S566A mutant may be acting in a dominant-negative manner to antagonize the endogenous action of B56δ. We further examined the PKC-dependent regulation of PP2A/B56δ using RNAi to down-regulate endogenous B56δ expression in N27 cells (Figure 5). B56δ-specific RNAi, expressed via adeno-associated virus (AAV) in N27 cells, resulted in reduced expression of endogenous B56δ protein by the B56δ-specific RNAi (less than 20% compared to control RNAi). Control RNAi expression had no effect compared to non-infected cells (not shown). Control or B56δ knock-down N27 cells were treated with DMSO vehicle, PMA or PMA together with rottlerin. PMA treatment resulted in dephosphorylation of phospho-Ser40 in control RNAi-infected cells, and this was inhibited by rottlerin. However, there was no effect of PMA treatment in cells in which B56δ expression was knocked down. Notably, the basal level of phospho-Ser40 was increased following knockdown of B56δ. Phosphorylation of Ser31 was unaffected under any of the conditions used.

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