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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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PKC activates PP2A/B56δ in vitro.FLAG-B56δ wt or FLAG-B56δ S566A mutant were expressed in N2a cells and active heterotrimeric complexes were immunoprecipitated from cell lysates using anti-FLAG antibody. Immunoprecipitated PP2A was mixed with purified PKC without (control) or with PKC activators (0.1 mg/ml phosphatidylserine, 0.02 mg/ml diacylglycerol, 0.2 mM CaCl2) and Mg ATP for 10 min. (A). Phosphorylation at Ser566 of B56δ was assessed by immunoblotting using anti-phospho-Ser566 antibody. As a loading control, total B56δ subunit and co-immunoprecipitated PP2A C subunit were assessed by immunoblotting. (B). Immobilized PP2A was suspended in phosphatase assay buffer then PP2A activity was measured using a phospho-peptide substrate (Promega, 5 min assay). Phosphatase activity is shown as means ± s.e.m. (n = 3). *, P<0.001, compared with no PKC activator control by student's t-test.
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pone-0026292-g003: PKC activates PP2A/B56δ in vitro.FLAG-B56δ wt or FLAG-B56δ S566A mutant were expressed in N2a cells and active heterotrimeric complexes were immunoprecipitated from cell lysates using anti-FLAG antibody. Immunoprecipitated PP2A was mixed with purified PKC without (control) or with PKC activators (0.1 mg/ml phosphatidylserine, 0.02 mg/ml diacylglycerol, 0.2 mM CaCl2) and Mg ATP for 10 min. (A). Phosphorylation at Ser566 of B56δ was assessed by immunoblotting using anti-phospho-Ser566 antibody. As a loading control, total B56δ subunit and co-immunoprecipitated PP2A C subunit were assessed by immunoblotting. (B). Immobilized PP2A was suspended in phosphatase assay buffer then PP2A activity was measured using a phospho-peptide substrate (Promega, 5 min assay). Phosphatase activity is shown as means ± s.e.m. (n = 3). *, P<0.001, compared with no PKC activator control by student's t-test.

Mentions: Our previous studies have shown that exogenous expression of the B56δ subunit results in formation of a heterotrimeric complex that can be isolated from cell lysates (Ahn et al. 2007a). Wild-type B56δ (FLAG-B56δ wt) or a mutant in which Ser566 was replaced by alanine (FLAG-B56δ S566A) were expressed in N2a cells, and PP2A heterotrimers were immunoprecipitated with anti-FLAG antibody. The immuno-purified PP2A preparations were then incubated in vitro with PKC and MgATP in the absence or presence of a PKC activator mixture which contained phosphatidylserine, diacylglycerol, and Ca2+ (Figure 3). Using immunoblotting, for the PP2A preparation that contained wild-type B56α subunit, a low level of phosphorylation at Ser566 was observed in the presence of PKC, and this was increased with addition of the PKC activator mixture (Figure 3A). However, as expected, only background phospho-Ser566 signal was detected in the PP2A preparation that contained B56δ with the S566A mutation.


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)

PKC activates PP2A/B56δ in vitro.FLAG-B56δ wt or FLAG-B56δ S566A mutant were expressed in N2a cells and active heterotrimeric complexes were immunoprecipitated from cell lysates using anti-FLAG antibody. Immunoprecipitated PP2A was mixed with purified PKC without (control) or with PKC activators (0.1 mg/ml phosphatidylserine, 0.02 mg/ml diacylglycerol, 0.2 mM CaCl2) and Mg ATP for 10 min. (A). Phosphorylation at Ser566 of B56δ was assessed by immunoblotting using anti-phospho-Ser566 antibody. As a loading control, total B56δ subunit and co-immunoprecipitated PP2A C subunit were assessed by immunoblotting. (B). Immobilized PP2A was suspended in phosphatase assay buffer then PP2A activity was measured using a phospho-peptide substrate (Promega, 5 min assay). Phosphatase activity is shown as means ± s.e.m. (n = 3). *, P<0.001, compared with no PKC activator control by student's t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198769&req=5

pone-0026292-g003: PKC activates PP2A/B56δ in vitro.FLAG-B56δ wt or FLAG-B56δ S566A mutant were expressed in N2a cells and active heterotrimeric complexes were immunoprecipitated from cell lysates using anti-FLAG antibody. Immunoprecipitated PP2A was mixed with purified PKC without (control) or with PKC activators (0.1 mg/ml phosphatidylserine, 0.02 mg/ml diacylglycerol, 0.2 mM CaCl2) and Mg ATP for 10 min. (A). Phosphorylation at Ser566 of B56δ was assessed by immunoblotting using anti-phospho-Ser566 antibody. As a loading control, total B56δ subunit and co-immunoprecipitated PP2A C subunit were assessed by immunoblotting. (B). Immobilized PP2A was suspended in phosphatase assay buffer then PP2A activity was measured using a phospho-peptide substrate (Promega, 5 min assay). Phosphatase activity is shown as means ± s.e.m. (n = 3). *, P<0.001, compared with no PKC activator control by student's t-test.
Mentions: Our previous studies have shown that exogenous expression of the B56δ subunit results in formation of a heterotrimeric complex that can be isolated from cell lysates (Ahn et al. 2007a). Wild-type B56δ (FLAG-B56δ wt) or a mutant in which Ser566 was replaced by alanine (FLAG-B56δ S566A) were expressed in N2a cells, and PP2A heterotrimers were immunoprecipitated with anti-FLAG antibody. The immuno-purified PP2A preparations were then incubated in vitro with PKC and MgATP in the absence or presence of a PKC activator mixture which contained phosphatidylserine, diacylglycerol, and Ca2+ (Figure 3). Using immunoblotting, for the PP2A preparation that contained wild-type B56α subunit, a low level of phosphorylation at Ser566 was observed in the presence of PKC, and this was increased with addition of the PKC activator mixture (Figure 3A). However, as expected, only background phospho-Ser566 signal was detected in the PP2A preparation that contained B56δ with the S566A mutation.

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