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

“α-Synuclein (α-Syn), a protein highly enriched in presynaptic terminals (Maroteaux et al., 1988), is implicated in normal brain function (George et al., 1989) as well as disease (reviewed by Perez and Hastings, 2004).”

“An active PP2A enzyme consists of a heterotrimer of the structural A subunit, a catalytic C subunit, and a regulatory B subunit (Dobrowsky et al., 1993).”

“The A and C subunits are ubiquitously expressed (Mayer et al., 1991) and form the catalytic complex (PP2A/C), which interacts with at least three different families of regulatory B subunits, as well as with certain tumor antigens (Mumby and Walter, 1993).”

“Control MN9D cells with low endogenous levels of α-Syn, included both untransfected parental MN9D cells and MN9D cells stably transfected with either pcDNA3 or GFP vector (Perez et al., 2002).”

“…protein 14-3-3 (Ostrerova et al., 1999), which binds to and regulates the activity of many cellular proteins. α-Syn interacts with and affects the activity of the enzymes phospholipase D (Ahn et al., 2002), protein kinase C and the extracellular regulated kinases (ERK) (Ostrerova et al., 1999).”

“Since it is well-documented that Ser40 dephosphorylation occurs almost exclusively by PP2A (Berresheim and Kuhn, 1994; Dunkley et al., 2004; Haavik et al., 1989; Leal et al., 2002) our findings indicate that (1) TH Ser40 is dephosphorylated by PP2A in dopaminergic cells, and (2) the Ser40 residue on TH remains accessi…”

“…kinase mediating Ser40 phosphorylation and because α-Syn is so strongly implicated in enzymatic inhibition, e.g. ERK2, PLD2, TH (Iwata et al., 2001; Jenco et al., 1998; Perez et al., 2002) we first hypothesized that the reduction in TH Ser40 phosphorylation was probably occurring by α-Syn…”

“Cell lysates prepared in 1% NP40 buffer containing protease inhibitors were sonicated for ∼5 seconds and particulates were eliminated by centrifugation at 15,000 g for 10 minute at 4°C as previously described (Perez et al., 1999).”

“…there is evidence that B subunits are associated with targeting the PP2A/C catalytic complexes to various intracellular sites such as microtubules (McCright et al., 1996; Sontag et al., 1995) and mitochondria (Ruvolo et al., 2002) suggesting that PP2A complexes are actively trafficked by their…”

“It is known that 14-3-3 can activate TH (Ichimura et al., 1988) by 14-3-3 binding first to TH Ser19 then to TH Ser40 (Kleppe et al., 2001).”

“a-Synuclein is quite promiscuous in its binding properties, and may bind various proteins in neuronal cells, including components of dopamine metabolism, such as tyrosine hydroxylase (TH) and the dopamine transporter (DAT), modulating their function (Ostrerova et al. 1999; Perez et al. 2002; Wersinger and Sidhu 2003;…”

“TBP-1 [332,333], phospholipase D [334], protein phosphatase 2A [335], DAT [336], mitochondrial complex IV enzyme cytochrome oxidase”

“-Synuclein inhibits dopamine synthesis by inhibiting the expression and activity of tyrosine hydroxylase [154, 168–170, 204], likely via reducing the phosphorylation state of tyrosine hydroxylase and stabilizing dephosphorylated inactive tyrosine hydroxylase [168, 205–207].”

“…on effects of the protein on catecholamine synthesis, uptake, and vesicular accumulation (Lee et al., 2001; Lotharius et al., 2002; Perez et al., 2002; Wersinger et al., 2003; Peng et al., 2005), is that amount of transmitter released per vesicle fusion event (quantal size) might be decreased.”