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First published online 19 July 2005
doi: 10.1242/jcs.02481

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-Synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells

Xiangmin M. Peng^1,*, Roya Tehranian^1,, Paula Dietrich^3,, Leonidas Stefanis^3,4,¶ and Ruth G. Perez^1,2,**

¹ Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
² Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
³ Department of Neurology, Columbia University, New York, NY, 10027-6902, USA
⁴ Department of Pathology, Columbia University, New York, NY, 10027-6902, USA

View larger version (31K): [in a new window]   Fig. 1. -Syn overexpression inhibits TH Ser40 phosphorylation. Representative immunoblots from a single experiments comparing 20 µg total protein from MN9D and PC12 cells. (Left column) (A) -Syn expression is low in UT (lane 1) and plasmid-transfected MN9D cells (lane 2), but increased >10-fold in stably transfected -Syn MN9D cells (lane 3). (B) Total TH protein is abundant in all MN9D cells, although, in this experiment, a reduction in total TH is apparent in the -Syn-overexpressing cells, although that was not always noted for -Syn MN9D cells. (C), P-Ser40 levels are virtually identical for UT (lane 1) and plasmid transfected cells (lane 2), but significantly reduced in the -Syn MN9D cells (lane 3). (Right column) Similar to MN9D cells (A) -Syn expression is low in UT (lane 1) and plasmid-transfected PC12 cells (lane 2), but increased 20-fold in stably transfected -Syn PC12 cells (lane 3) at 72 hours after induction. (B) Total TH protein is equally abundant in all PC12 cells regardless of -Syn levels. (C) P-Ser40 levels are virtually identical for control PC12 cells (UT, lane 1; plasmid transfected PC12 cells, lane 2) but significantly reduced in the -Syn Pc12 cells (lane 3). (D) P-Ser40 data from several experiments were normalized to total TH and expressed as percentage relative to control UT MN9D (left) and PC12 (right) cells. Untransfected cells (UT), plasmid-transfected control cells (plasmid), and stably transfected -Syn-overexpressing cells (-Syn). Values are mean ± s.e.m. from three independent experiments for each cell line. ***P<0.001.

View larger version (17K): [in a new window]   Fig. 2. -Syn overexpression does not alter PP2A protein levels in MN9D or PC12 cells. (Top) A representative immunoblot for MN9D and PC12 cells reacted with the PP2A antibody. (Below) Bar graph showing that UT, plasmid and -Syn MN9D and PC12 cells had essentially identical PP2A levels. Untransfected cells (UT), plasmid-transfected control cells (plasmid), and stably transfected -Syn-overexpressing cells (-Syn). Data are expressed as the mean percent change relative to UT cells ± s.e.m. of combined data from three independent experiments.

View larger version (20K): [in a new window]   Fig. 3. -Syn contributes to PP2A activation in MN9D and PC12 cells. (A) Cell lysates from MN9D cells and from induced PC12 cells were immunoprecipitated for the PP2A catalytic subunit. A representative immunoblot shows equivalent amounts of PP2A immunoprecipitated from all samples. (B) The activity of the immunoprecipitated PP2A was measured for MN9D cells (gray bars) and for induced PC12 cells (black bars). Plasmid, plasmid-transfected control cells; -Syn; stably transfected -Syn-overexpressing cells. Values are mean ± s.e.m. from three independent experiments. *P<0.05.

View larger version (28K): [in a new window]   Fig. 4. Interaction of -Syn and PP2A in cells and in rat brain. (A) -Syn PC12 cell lysates were co-immunoprecipitated and proteins were separated by SDS-PAGE and the western blots (WB) were reacted with -Syn antibody (-S WB) or PP2A antibody (PP2A WB). Left blot shows the levels of -Syn in cells from the initial homogenate (lane 1), in the homogenate after co-immunoprecipitation (co-IP; lane 2), and -Syn immunoprecipitated with the Syn-1 antibody (lane 3). Right blot in A was prepared from the same co-IP sample with the levels of PP2A in the initial homogenate (lane 1), PP2A in the homogenate after co-IP (lane 2), and PP2A co-immunoprecipitated along with -Syn using the Syn-1 antibody (lane 3). Non-specific bands, two of which appear to be IgG bands (asterisks) are evident in both blots. (B) The -Syn WB reveals -Syn immunoprecipitated from rat striatum (left lane) and in the PP2A WB the PP2A that co-immunoprecipitated with -Syn using the Syn-1 antibody (right lane). Relative molecular mass (Mr, x10-3) was determined from prestained standards. -S WB, -synuclein Syn-1 antibody-reacted western blot; PP2A WB, PP2A antibody-reacted western blot.

View larger version (22K): [in a new window]   Fig. 5. Blockade of PP2A activity with okadaic acid demonstrates a role for PP2A in Ser40 dephosphorylation in MN9D and PC12 cells. PP2A inhibition by 10 nM-1.0 µM okadaic acid for 1 hour, caused an increase in P-Ser40 levels in all conditions when compared to parallel vehicle-treated controls for each condition. The baseline value was set to zero to demonstrate the fold increase in P-Ser40 levels above baseline for each condition. (A) Okadaic acid at low doses (0-100 nM) produced small changes in P-Ser40 in plasmid control MN9D cells but large significant increases in P-Ser40 in -Syn-overexpressing MN9D cells. Vehicle-treated cells were essentially unchanged from baseline at these concentrations of okadaic acid. (B) With higher dose okadaic acid, even more robust increases in P-Ser40 were noted for -Syn MN9D and induced -Syn PC12 cells, suggesting that low baseline P-Ser40 phosphorylation levels in -Syn overexpressors had probably occurred through effects on PP2A activation. Values are mean ± s.e.m. of two to six independent experiments. *P<0.01.

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