Limits...
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.

Show MeSH

Related in: MedlinePlus

The PKC activator PMA increases phosphorylation of B56δ at Ser566.(A) N2a cells expressing FLAG-B56δ subunit were treated with DMSO (vehicle) or PMA (5 nM) for 5 min. Cells were lysed and proteins were analyzed by SDS-PAGE and immunoblotting with phospho-specific antibodies to the indicated sites in B56δ. Total B56δ was analyzed with anti-FLAG antibody. Upper panels show immunoblots. The bar graph shows quantification of immunoblot data normalized in each experiment to vehicle for each site as means ± s.e.m. (n = 3). *, P<0.001 compared with vehicle-treated control by student's t-test. (B) N2a cells expressing FLAG-B56δ were treated with different concentrations of PMA (0, 1, 10, 100, 1000 nM) for 5 min. Phosphorylation of Ser566 and total B56δ was assayed as in (A). The bar graph shows quantification of data normalized in each experiment to the zero PMA condition as means ± s.e.m. (n = 3). *, P<0.05, **, P<0.01 compared with vehicle-treated control by student's t-test. (C) N2a cells expressing FLAG-B56δ were treated with 5 nM PMA for the indicated times. Phosphorylation of Ser566 and total B56δ was assayed as in (A). The bar graph shows quantification of data normalized in each experiment to the zero time condition as means ± s.e.m. (n = 3). *, P<0.01, **, P<0.001 compared with vehicle treated control by student's t-test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198769&req=5

pone-0026292-g001: The PKC activator PMA increases phosphorylation of B56δ at Ser566.(A) N2a cells expressing FLAG-B56δ subunit were treated with DMSO (vehicle) or PMA (5 nM) for 5 min. Cells were lysed and proteins were analyzed by SDS-PAGE and immunoblotting with phospho-specific antibodies to the indicated sites in B56δ. Total B56δ was analyzed with anti-FLAG antibody. Upper panels show immunoblots. The bar graph shows quantification of immunoblot data normalized in each experiment to vehicle for each site as means ± s.e.m. (n = 3). *, P<0.001 compared with vehicle-treated control by student's t-test. (B) N2a cells expressing FLAG-B56δ were treated with different concentrations of PMA (0, 1, 10, 100, 1000 nM) for 5 min. Phosphorylation of Ser566 and total B56δ was assayed as in (A). The bar graph shows quantification of data normalized in each experiment to the zero PMA condition as means ± s.e.m. (n = 3). *, P<0.05, **, P<0.01 compared with vehicle-treated control by student's t-test. (C) N2a cells expressing FLAG-B56δ were treated with 5 nM PMA for the indicated times. Phosphorylation of Ser566 and total B56δ was assayed as in (A). The bar graph shows quantification of data normalized in each experiment to the zero time condition as means ± s.e.m. (n = 3). *, P<0.01, **, P<0.001 compared with vehicle treated control by student's t-test.

Mentions: To initially address the ability of PKC to regulate the phosphorylation of the B56δ subunit, we expressed FLAG-B56δ in N2a cells, then treated cells with vehicle or the phorbol ester PMA under a variety of conditions (Figure 1). The phosphorylation level at four sites in B56δ (Ser53, Ser68, Ser81, Ser566) was analyzed with their respective phospho-specific antibody. PMA treatment increased the phosphorylation level of Ser566 ∼1.5-fold compared to vehicle-treated cells (Figure 1a). Exposure of N2a cells to different concentrations of PMA resulted in a dose responsive increase in Ser566 phosphorylation up to 10 nM (Figure 1b). In response to treatment with PMA, phosphorylation of B56δ was rapid and maximal after 30 min (Figure 1c). In the presence of 5 nM PMA, the phosphorylation at Ser566 increased ∼3.5-fold after 30 min compared to the vehicle-treated control.


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)

The PKC activator PMA increases phosphorylation of B56δ at Ser566.(A) N2a cells expressing FLAG-B56δ subunit were treated with DMSO (vehicle) or PMA (5 nM) for 5 min. Cells were lysed and proteins were analyzed by SDS-PAGE and immunoblotting with phospho-specific antibodies to the indicated sites in B56δ. Total B56δ was analyzed with anti-FLAG antibody. Upper panels show immunoblots. The bar graph shows quantification of immunoblot data normalized in each experiment to vehicle for each site as means ± s.e.m. (n = 3). *, P<0.001 compared with vehicle-treated control by student's t-test. (B) N2a cells expressing FLAG-B56δ were treated with different concentrations of PMA (0, 1, 10, 100, 1000 nM) for 5 min. Phosphorylation of Ser566 and total B56δ was assayed as in (A). The bar graph shows quantification of data normalized in each experiment to the zero PMA condition as means ± s.e.m. (n = 3). *, P<0.05, **, P<0.01 compared with vehicle-treated control by student's t-test. (C) N2a cells expressing FLAG-B56δ were treated with 5 nM PMA for the indicated times. Phosphorylation of Ser566 and total B56δ was assayed as in (A). The bar graph shows quantification of data normalized in each experiment to the zero time condition as means ± s.e.m. (n = 3). *, P<0.01, **, P<0.001 compared with vehicle treated control by student's t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026292-g001: The PKC activator PMA increases phosphorylation of B56δ at Ser566.(A) N2a cells expressing FLAG-B56δ subunit were treated with DMSO (vehicle) or PMA (5 nM) for 5 min. Cells were lysed and proteins were analyzed by SDS-PAGE and immunoblotting with phospho-specific antibodies to the indicated sites in B56δ. Total B56δ was analyzed with anti-FLAG antibody. Upper panels show immunoblots. The bar graph shows quantification of immunoblot data normalized in each experiment to vehicle for each site as means ± s.e.m. (n = 3). *, P<0.001 compared with vehicle-treated control by student's t-test. (B) N2a cells expressing FLAG-B56δ were treated with different concentrations of PMA (0, 1, 10, 100, 1000 nM) for 5 min. Phosphorylation of Ser566 and total B56δ was assayed as in (A). The bar graph shows quantification of data normalized in each experiment to the zero PMA condition as means ± s.e.m. (n = 3). *, P<0.05, **, P<0.01 compared with vehicle-treated control by student's t-test. (C) N2a cells expressing FLAG-B56δ were treated with 5 nM PMA for the indicated times. Phosphorylation of Ser566 and total B56δ was assayed as in (A). The bar graph shows quantification of data normalized in each experiment to the zero time condition as means ± s.e.m. (n = 3). *, P<0.01, **, P<0.001 compared with vehicle treated control by student's t-test.
Mentions: To initially address the ability of PKC to regulate the phosphorylation of the B56δ subunit, we expressed FLAG-B56δ in N2a cells, then treated cells with vehicle or the phorbol ester PMA under a variety of conditions (Figure 1). The phosphorylation level at four sites in B56δ (Ser53, Ser68, Ser81, Ser566) was analyzed with their respective phospho-specific antibody. PMA treatment increased the phosphorylation level of Ser566 ∼1.5-fold compared to vehicle-treated cells (Figure 1a). Exposure of N2a cells to different concentrations of PMA resulted in a dose responsive increase in Ser566 phosphorylation up to 10 nM (Figure 1b). In response to treatment with PMA, phosphorylation of B56δ was rapid and maximal after 30 min (Figure 1c). In the presence of 5 nM PMA, the phosphorylation at Ser566 increased ∼3.5-fold after 30 min compared to the vehicle-treated control.

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