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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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Dopamine synthesis in N27 cells is increased following down-regulation of B56δ.N27 cells were infected with AAV-control RNAi or AAV- B56δ RNAi for 72 h. Dopamine was measured in cell lysates by LC/MS with tritium labeled DA as a standard. Data are shown as means ± s.e.m. (n = 3). *, P<0.01 compared with control RNAi infected cells by student's t-test.
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pone-0026292-g006: Dopamine synthesis in N27 cells is increased following down-regulation of B56δ.N27 cells were infected with AAV-control RNAi or AAV- B56δ RNAi for 72 h. Dopamine was measured in cell lysates by LC/MS with tritium labeled DA as a standard. Data are shown as means ± s.e.m. (n = 3). *, P<0.01 compared with control RNAi infected cells by student's t-test.

Mentions: Previous studies have indicated that PKCδ-deficient N27 cells showed an increase in dopamine synthesis through enhanced TH activity [1]. We further examined the physiological role of TH regulation by PKC-mediated control of the B56δ subunit by measuring dopamine synthesis in N27 cells (Figure 6). Expression of B56δ was knocked down in N27 cells using AAV-mediated expression of B56δ RNAi. After 72 h, cells were harvested, and dopamine production was analyzed using LC/MS. N27 cells with reduced B56δ expression showed significantly increased dopamine levels compared to cells expressing a control RNAi plasmid. Dopamine levels were determined to be 1.59±0.16 µg/mg of protein in control RNAi cells compared to 3.69±0.21 µg/mg protein in B56δ RNAi expressing 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)

Dopamine synthesis in N27 cells is increased following down-regulation of B56δ.N27 cells were infected with AAV-control RNAi or AAV- B56δ RNAi for 72 h. Dopamine was measured in cell lysates by LC/MS with tritium labeled DA as a standard. Data are shown as means ± s.e.m. (n = 3). *, P<0.01 compared with control RNAi infected cells by student's t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026292-g006: Dopamine synthesis in N27 cells is increased following down-regulation of B56δ.N27 cells were infected with AAV-control RNAi or AAV- B56δ RNAi for 72 h. Dopamine was measured in cell lysates by LC/MS with tritium labeled DA as a standard. Data are shown as means ± s.e.m. (n = 3). *, P<0.01 compared with control RNAi infected cells by student's t-test.
Mentions: Previous studies have indicated that PKCδ-deficient N27 cells showed an increase in dopamine synthesis through enhanced TH activity [1]. We further examined the physiological role of TH regulation by PKC-mediated control of the B56δ subunit by measuring dopamine synthesis in N27 cells (Figure 6). Expression of B56δ was knocked down in N27 cells using AAV-mediated expression of B56δ RNAi. After 72 h, cells were harvested, and dopamine production was analyzed using LC/MS. N27 cells with reduced B56δ expression showed significantly increased dopamine levels compared to cells expressing a control RNAi plasmid. Dopamine levels were determined to be 1.59±0.16 µg/mg of protein in control RNAi cells compared to 3.69±0.21 µg/mg protein in B56δ RNAi expressing 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