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).”

“…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 associated interacting…”

“, 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.”

“…S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al., 1986; Wu et al., 1992).”

“The regulation of TH activity in the short term depends on the phosphorylation of key seryl residues (Ser19, Ser31 and Ser40) in the amino-terminal regulatory domain (Campbell et al., 1986; Haycock, 1990).”

“The regulatory B subunits of PP2A are known to be temporally expressed during development (Csortos et al., 1996; Mayer et al., 1991; McCright and Virshup, 1995; Ruediger et al., 1991) and neuron-specific isoforms have also been identified (Mayer et al.”

“Jo ur na l o f C el l S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.,…”

“…PP2A may affect PP2A conformation or trafficking and subsequently contribute to its activation. α-Syn oxidative modification or aggregation occurs in neurodegenerative diseases such as Alzheimer’s disease, a condition in which PP2A is also implicated (Trojanowski and Lee, 1995; Zhao et al., 2003).”

“, 1991) and neuron-specific isoforms have also been identified (Mayer et al., 1991).”

“The MN9D dopaminergic cells express abundant TH (Choi et al., 1991) and synthesize DA (Choi et al.”

“Jo ur na l o f C el l S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.,…”

“The substrate specificity of PP2A appears to be determined by the regulatory B subunits (Cegielska et al., 1994; Csortos et al., 1996) and there is evidence that B subunits are associated with targeting the PP2A/C catalytic complexes to various intracellular sites such as microtubules (McCright et…”

“, 1995) and mitochondria (Ruvolo et al., 2002) suggesting that PP2A complexes are actively trafficked by their associated interacting proteins, one of which may be α-Syn.”

“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 inhibition of PKA.”

“…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).”

“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).”

“, 2002), which reduces TH activity both in dopaminergic cells (Haavik et al., 1989) and in rat striatum (Bevilaqua et al.”

“Of these, phosphorylated Ser40 is a major contributor to DA synthesis in vivo (Ramsey et al., 1996).”

“These constructs form the basis of the ‘bomb system’ for inducible transgene expression (Suhr et al., 1998; Suhr et al., 2001) and were generously provided by Fred Gage and Steve Suhr at the Salk Institute (La Jolla, CA, USA).”

“The MN9D dopaminergic cells express abundant TH (Choi et al., 1991) and synthesize DA (Choi et al., 1992).”

“Jo ur na l o f C el l S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.,…”

“The regulatory B subunits of PP2A are known to be temporally expressed during development (Csortos et al., 1996; Mayer et al., 1991; McCright and Virshup, 1995; Ruediger et al., 1991) and neuron-specific isoforms have also been identified (Mayer et al.”

“Generation of stably transfected inducible PC12 cell lines α-Syn was cloned into pcDNA3 as previously described (Stefanis et al., 2001) followed by subcloning into the SKSP shuttle cloning vector using HindIII-XhoI.”

“Low-passage number MN9D cells were grown as previously described (Ugarte et al., 2003).”

“2). α-Syn increases PP2A activity and binds to the PP2A catalytic domain We measured PP2A activity from the MN9D and induced PC12 cells using a well-established immunoprecipitation and phosphatase activity protocol (Begum and Ragolia, 1996).”

“Although multiple kinases can phosphorylate TH on Ser40 in vitro, including cyclic GMP-dependent protein kinase (Rodriguez-Pascual et al., 1999; Roskoski et al., 1987), CamKII (Campbell et al., 1986; Yamauchi and Fujisawa, 1981), 3523 α-Synuclein is an abundant presynaptic protein implicated in…”

“There is additional evidence that the binding of 14-3-3 to TH phosphorylated on Ser19 and Ser40 stabilizes its conformation and enhances TH activity (Bevilaqua et al., 2001).”

“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).”

“Furthermore, we treated cells with low to high dose okadaic acid (5 nM-1 μM) dissolved in DMSO (0.13 μM) (Garcia et al., 2002; Haavik et al., 1989) for 1 hour to assess the impact on PP2A inhibition in the presence of α-Syn overexpression and saw a similar effect.”

“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).”

“, 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al., 1986; Wu et al., 1992).”

“…PP2A may affect PP2A conformation or trafficking and subsequently contribute to its activation. α-Syn oxidative modification or aggregation occurs in neurodegenerative diseases such as Alzheimer’s disease, a condition in which PP2A is also implicated (Trojanowski and Lee, 1995; Zhao et al., 2003).”

“…Ser40 in vitro, including cyclic GMP-dependent protein kinase (Rodriguez-Pascual et al., 1999; Roskoski et al., 1987), CamKII (Campbell et al., 1986; Yamauchi and Fujisawa, 1981), 3523 α-Synuclein is an abundant presynaptic protein implicated in neuronal plasticity and neurodegenerative diseases.”

“It is well documented that dephosphorylation of TH Ser40 occurs almost exclusively by the phosphatase PP2A (Berresheim and Kuhn, 1994; Haavik et al., 1989; Leal et al., 2002), which reduces TH activity both in dopaminergic cells (Haavik et al.”

“Tebufenozide, which is a molt-inducing insecticide that mimics the action of ecdysone (Addison, 1996), was used for induction of α-Syn expression.”

“It is well documented that dephosphorylation of TH Ser40 occurs almost exclusively by the phosphatase PP2A (Berresheim and Kuhn, 1994; Haavik et al., 1989; Leal et al., 2002), which reduces TH activity both in dopaminergic cells (Haavik et al., 1989) and in rat striatum (Bevilaqua et al., 2003).”

“Although multiple kinases can phosphorylate TH on Ser40 in vitro, including cyclic GMP-dependent protein kinase (Rodriguez-Pascual et al., 1999; Roskoski et al., 1987), CamKII (Campbell et al., 1986; Yamauchi and Fujisawa, 1981), 3523 α-Synuclein is an abundant presynaptic protein implicated in…”

“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…”

“Jo ur na l o f C el l S ci en ce 3524 PKC (Albert et al., 1984; Vulliet et al., 1985), MSK-1 (Toska et al., 2002), MAPKAP kinases 1 and 2 (Sutherland et al., 1993), it is the cAMP-dependent protein kinase PKA (Joh et al., 1978) that predominantly phosphorylates TH Ser40 in vivo (Campbell et al.,…”

“Membrane bound PP2A in brain is reportedly less active (Sim et al., 1998), thus the interaction of PP2A with α-Syn within the cytosol may serve to stimulate PP2A activity.”

“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.”