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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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Rottlerin inhibits PMA-dependent phosphorylation of Ser566 in B56δ.N2a cells expressing FLAG-B56δ were pretreated without or with rottlerin (5 µM) for 30 min as indicated and then treated with DMSO (Control) or PMA (10 nM) for an additional 10 min. Phosphorylation of Ser566 and total B56δ was assayed as above. The bar graph shows quantification of immunoblot data (not shown) normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001, **, P<0.01 compared with vehicle-treated vector control by one-way ANOVA with Newman-Keuls multiple comparison test.
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pone-0026292-g002: Rottlerin inhibits PMA-dependent phosphorylation of Ser566 in B56δ.N2a cells expressing FLAG-B56δ were pretreated without or with rottlerin (5 µM) for 30 min as indicated and then treated with DMSO (Control) or PMA (10 nM) for an additional 10 min. Phosphorylation of Ser566 and total B56δ was assayed as above. The bar graph shows quantification of immunoblot data (not shown) normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001, **, P<0.01 compared with vehicle-treated vector control by one-way ANOVA with Newman-Keuls multiple comparison test.

Mentions: We next investigated the type of PKC isozyme involved in B56δ phosphorylation. FLAG-B56δ was expressed in N2a cells, and the effect of the PKCδ inhibitor rottlerin was examined (Figure 2). Cells were pre-incubated in the absence or presence of rottlerin for 30 min, treated with DMSO vehicle or PMA. PMA-dependent phosphorylation at Ser566 was inhibited by rottlerin. Rottlerin alone did not have any significant effect on Ser566 phosphorylation. Go6976, a PKCα specific inhibitor, did not affect Ser566 phosphorylation in a statistically significant manner (data not shown) suggesting that PKCδ is likely a major PKC isozyme involved in B56δ phosphorylation in N2a cells.


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)

Rottlerin inhibits PMA-dependent phosphorylation of Ser566 in B56δ.N2a cells expressing FLAG-B56δ were pretreated without or with rottlerin (5 µM) for 30 min as indicated and then treated with DMSO (Control) or PMA (10 nM) for an additional 10 min. Phosphorylation of Ser566 and total B56δ was assayed as above. The bar graph shows quantification of immunoblot data (not shown) normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001, **, P<0.01 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-g002: Rottlerin inhibits PMA-dependent phosphorylation of Ser566 in B56δ.N2a cells expressing FLAG-B56δ were pretreated without or with rottlerin (5 µM) for 30 min as indicated and then treated with DMSO (Control) or PMA (10 nM) for an additional 10 min. Phosphorylation of Ser566 and total B56δ was assayed as above. The bar graph shows quantification of immunoblot data (not shown) normalized in each experiment to control as means ± s.e.m. (n = 3). *, P<0.001, **, P<0.01 compared with vehicle-treated vector control by one-way ANOVA with Newman-Keuls multiple comparison test.
Mentions: We next investigated the type of PKC isozyme involved in B56δ phosphorylation. FLAG-B56δ was expressed in N2a cells, and the effect of the PKCδ inhibitor rottlerin was examined (Figure 2). Cells were pre-incubated in the absence or presence of rottlerin for 30 min, treated with DMSO vehicle or PMA. PMA-dependent phosphorylation at Ser566 was inhibited by rottlerin. Rottlerin alone did not have any significant effect on Ser566 phosphorylation. Go6976, a PKCα specific inhibitor, did not affect Ser566 phosphorylation in a statistically significant manner (data not shown) suggesting that PKCδ is likely a major PKC isozyme involved in B56δ phosphorylation in N2a cells.

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