Christof Rickert Seminar Softskills, 28.05.2010

Christof Rickert
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Seminar Softskills, 28.05.2010

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ABSTRACT
BACKGROUND
PHOSPHOPROTEIN PHOSPHATASE 2A CATALYZES THE DEPHOSPHORYLATION
OF pSer129 ALPHA-‐SYNUCLEIN
Jeffry. B. Stock 1, 2 , Xuyan Feng 2 , Yang Chao 2 , Walter Chen 1 , Neelanjana Ray 2 , Tsuyoshi Ishii 1,2 , Jose R. Fernandez 2 , Yi Liu 2 ,
Arvind Raghavan 2 , Maxwell Stock 2 , M. Maral Mouradian 3
1 Dept. of Molecular Biology, Princeton University, Princeton, NJ, USA. 2 Signum Biosciences, Monmouth Junction, NJ, USA. 3 Dept. Neurology, UMDNJ-RWJMS, Piscataway, NJ, USA
Modulation of phosphatase activity is a promising approach for
the development of therapeutics targeting hyperphosphorylated
proteins in neurodegenerative diseases. (See Poster # 238.6/G7;;
�ENHANCING PROTEIN PHOSPHATASE 2A ACTIVITY IS
NEUROPROTECTIVE IN MOUSE MODELS OF �����������
DISEASE�� .
Phosphoprotein phosphatase 2A (PP2A)
� Multi-subunit protein, accounts for over 50% of brain Ser/Thr
phosphatase activity
� Dimeric core enzyme composed of 65-kDa scaffold A-subunit &
36-kDa catalytic C-subunit.
� Various B subunits serve to regulate PP2A specificity1 .
� Reversible carboxyl-methylation at C-terminal Leu309 of the Csubunit,
increases affinity of B subunit to AC dimers2 A pathological hallmark of Parkinson¹s Disease (PD) is the presence
AB C form of PP2A
of Lewy bodies (LBs) in surviving neurons whose major components � Accounts for over 70% of tau phosphatase activity in vivo
include -synuclein ( -Syn). Phosphorylation of -Syn at serine 129
(S129) facilitates its aggregation, and -Syn in LBs tends to be
phosphorylated at S129. Several kinases that phosphorylate -Syn
at S129 have been characterized, but the counteracting
phosphatase(s) have not been identified. Here we show that protein
phosphatase 2A (PP2A) is the major phosphatase that
dephosphorylates S129 in an SH-SY5Y-derived neuroblastoma cell
line that overproduces -Syn. This conclusion is supported by
biochemical assays of P-S129 -Syn dephosphorylation using a
variety of different purified and reconstituted phosphatase enzymes.
In contrast to results obtained with PP2A, other major
serine/threonine phosphatases including PP1, PP2B, and PP2C
exhibit much lower activity toward P-S129 -Syn. PP2A activity is
regulated by carboxyl methylation of the phosphatase catalytic
subunit and our in vitro results indicate that PP2A methylation can
act to enhance phosphatase activity toward P-S129 -Syn. These RESULTS
findings raise the possibility that dysregulation of PP2A could
contribute to -syn aggregation and to the pathogenesis of PD.
-->
assembly of AB C heterotrimers (Figure 1).
4
� Tau mutations associated with FTDP-17 decrease affinity of PP2A
for tau, in vivo5 � Loss of PP2A methylation and activity reported in brains of
patients with �����������
disease and ������ syndrome6,7 .
A greater understanding of phosphatase specificities
towards -synuclein is required to address the possible role
of PP2A dysregulation in the etiology of ����������� disease
and related synucleinopathies.
4 Sontag E et. al., Neuron (1996), 17(6): 1201-1207.
5 Goedert M et. al., J Neurochem. (2000), 75(5): 2155-2162.
6 Gong CX & Iqbal K. Curr Med Chem. (2008), 15(23): 2321-2328.
7 Sontag E et. al., J Neuropathol Exp Neurol. 2004), 63(10): 1080-1091.
PP2A dephosphorylates pS129 -synuclein in SH-SY5Y cells.
� Varying concentrations of broad-specificity phosphatase inhibitor
okadaic acid (OA) and the PP1-specific inhibitor tautomycetin
(Tc), were used to investigate relative activities of the two
phosphatases in -synuclein over-expressing SH-SY5Y (SH-
SY5Y/syn) cells.
� 250 nM okadaic acid (OA) was used to specifically inhibit PP2A in
SH-SY5Y/syn cells. Since PP1 activity is not completely abolished
at this concentration of OA, extracts were further treated with 35
nM tautomycetin (Tc) and tested for PP2A activity (Fig. 2A).
� SH-SY5Y/syn cells were also treated with 250 nM OA for 2h, and
extracts were further treated with 2 M OA to test for PP1
activity (Fig. 2B).
� PP2B is inhibited only at much higher concentrations of OA (IC50 ~4�M) and PP2C is virtually insensitive to OA.
� OA-treated extracts failed to dephosphorylate pS129 -synuclein,
while Tc-treated extracts showed little or no effect on pS129 -
syn (Fig. 2C).
A B
ight protected. F1000 Poster Bank. Copyright protected. F1000 Poster Bank. Copyright protecte
� Methylation catalyzed by a PP2A-specific, S-adenosyl methionine
(SAM)-dependent methyltransferase (PPMT)
� Demethylation is mediated by a PP2A-specific methylesterase
(PME-1) 3 (Figure 1).
Figure 1
Scaffold (A) subunit
Catalytic (C) subunit
PME-1
(-) CH3
AC core dimer
(+) CH3
Regulatory (B) subunit
1
Lechward et. al., Acta Biochim. Pol. (2001), 48(4): 921-933.
2
Tolstykh L et. al., EMBO J. (2000), 19(21): 5682-5691.
3
Lee J & Stock JB. J Biol. Chem. (1993), 268(26): 19192-19195.
PPMT
PP2A holoenzyme
Figure 2. PP2A dephosphorylates pS129 -­‐syn in SH-­‐SY5YA cells. (A) SH-­‐SY5Y/syn cells
were treated with EtOH (Ctl) or 250 nM okadaic acid (OA) for 1 h. Ctl and OA-­‐treated
cell extracts were then incubated with DMSO or 35 nM Tc (to completely inhibit PP1)
respectively. Inhibitor-­‐treated extracts were assayed for PP2A activity (defined as the
fraction of activity resistant to 35 nM Tc but inhibited by 5 nM OA), using 32P-­‐labelled phosphorylase a. Activity is expressed as a percentage of PP2A activity in Ctl cells. *P <
0.01 by ��������� t-­‐test. Data are mean ± SD. (B) SH-­‐SY5Y/syn cells were treated with
EtOH (Ctl) or OA (250 nM) for 2 h. Extracts were incubated with 5 nM OA or 2 ��OA
prior to PP1 activity assay (defined as the fraction of activity resistant to 5 nM OA but
inhibited by 2 ��OA), using 32P-­‐labeled phosphorylase a. Activity is expressed as a
percentage of PP1 activity in Ctl-­‐treated cells. Data are mean±SD of duplicate assays,
representative of three experiments. (C) SH-­‐SY5Y/syn cells were treated with EtOH
(Ctl) or 250 nM okadaic (OA) for 1h. Extracts were analyzed by Western blotting using
antibodies against pS129 -­‐synuclein and total -­‐synuclein (Abcam).
Copyright protected. F1000 Poster Bank. Copyright protected. F1000 Poster Bank. Copyright protected. F1000 Pos
C
PP1 is not the primary pS129 -synuclein phosphatase in
neuroblastoma cells.
� PP2A and PP1 constitute approximately 90% of the total Ser/Thr
phosphatase activity in mammalian cells.
� We treated SH-SY5Y/syn cells with 1 M tautomycetin (Tc), a PP1specific
inhibitor. Cell extracts were then treated with varying
concentrations of okadaic acid (OA) to investigate relative
activities of the two enzymes in these cells (Fig. 3A).
� Tc-treated lysates did not affect pS129 -syn (Fig. 3B), indicating
that PP1 does not dephosphorylate -synuclein at this residue.
Figure 3. Evaluation of PP1 & PP2A activities in neuroblastoma cells. SH-­‐SY5Y/syn
cells were treated with DMSO (Ctl) or 1 M tautomycetin (Tc) for 20 min. (A) Cell
extracts were then either untreated, or incubated with 5 nM and 2 M okadaic acid,
to define PP2A, PP1 and residual phosphatase activities respectively. Inhibitor-­‐treated
extracts were assayed for phosphatase activity using 32P-­‐labelled phosphorylase a. *P
����������������������
-­‐test. All data shown are mean ± S.D. of duplicate assays and are
representative of three experiments. (B) SH-­‐SY5Y/syn cells were treated with DMSO
(Ctl) or tautomycetin (Tc) for 20 min. (right panels). Extracts were analyzed by Western
blotting using antibodies against pS129 -­‐synuclein and total -­‐synuclein (Abcam).
PP2A, but not PP1, dephosphorylates pS129 -syn in vitro.
Figure 4. In vitro dephosphorylation of pS129 -­‐synuclein. Purified human -­‐
synuclein was phosphorylated in vitro with CK2 (NEB). Purified, unmethylated and
reconstituted PP2A (AC-­‐dimer), and purified PP1 were incubated at the indicated
concentrations with phospho-­‐ -­‐syn for 30 min. at 30 o C. Reactions were analyzed by
Western blotting using pS129 -­‐synuclein (pS129 -­‐syn) antibodies (Abcam).
PP2A activities towards phosphorylated -synuclein are
dependent on methylation and subunit composition.
Methylation of the AC dimer (Fig. 5A) enhances PP2A activity
towards pS129 -synuclein.
A
PP1
PP2A
PP1 Activity (5nM OA-insensitive,
2 M OA-inhibited)
PP2A Activity (5nM OA-inhibited)
Residual Activity (2 M OA-insensitive)
0 2 20 200 (nM)
Figure 5. Differential PP2A activities towards pS129 -­‐synuclein. (A) Phosphorylated
-­‐synuclein was prepared as described in Figure 4. Unmethylated or in vitro methylated
PP2A AC-­‐dimers were incubated at the indicated concentrations for 30 min. at 30 o C with
phospho-­‐ -­‐synuclein, reactions were analyzed by Western blotting using pS129 -­‐
synuclein (pS129 -­‐syn) antibodies (Abcam).
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A
B
Figure 5.
Methylation of the trimeric holoenzyme (Fig. 5B), enhances PP2A
activity towards pS129 -synuclein.
B PP2A 0 12 25 50 100 200 nM
The trimeric holoenzyme containing the B -subunit (AB C)
dephosphorylates pS129 -syn more efficiently than holoenzymes
containing the ��(���C), ������ C), or ��� ������� holoenzymes.
C
Demeth-AC
Demeth-AB�C
Meth-AB�C
PP2A 0 12 25 50 100 200 nM
AB�C
����C
���1C
�����
Figure 5. Methylation-­‐dependent phosphatase activities towards phospho-­‐synuclein.
Phosphorylated -­‐synuclein was prepared as in Figure 4. Purified, methylated and
reconstituted PP2A enzymes at the indicated concentrations were incubated with
phospho-­‐ -­‐synuclein for 30 min. at 30 o C. Reactions were analyzed by Western blotting
with pS129 -­‐syn and Total -­‐syn antibodies (Abcam). (B) Demeth-­‐AC or Meth-­‐AC
dimers were reconstituted with B subunit to yield Demeth-­‐AB C and Meth-­‐AB C
holoenzymes respectively. (C) Demeth-­‐AC was reconstituted in vitro with B , �� , ��
and ���subunits to yield the AB C, ��� C, ��� C, and �����holoenzymes respectively.
,
SIG1012, a small-molecule PME-1 inhibitor, reduces PP2A
demethylation and pS129 -syn levels in SH-SY5Y/syn cells.
% of untreated
120
100
Figure 6. SIG1012 reduces PP2A demethylation and phospho-­‐syn levels in cells. SH-­‐
SY5Y/syn cells were incubated with the indicated SIG1012 concentrations for 4 h.
Lysates were analyzed by Western blotting using antibodies to PP2A demethylated C-­‐
subunit (Demeth-­‐C; Millipore) and total C-­‐subunit (Total-­‐C), pS129 -­‐syn (p-­‐syn) and
total -­‐syn (Abcam). Signal intensities were normalized to respective loading controls,
data are average normalized intensities from 3 independent experiments; * p < 0.05.
CONCLUSION
80
60
40
20
0
0 uM SIG1012 5 uM SIG1012 25 uM SIG1012
*
*
Demeth-C/Total-C p-syn/Total-syn
� In SH-SY5Y/syn cells, -synuclein is dephosphorylated primarily by
PP2A, not PP1.
� Biochemically reconstituted AB C PP2A holoenzymes dephosphorylate
-synuclein more efficiently than other trimeric forms.
� Dephosphorylation of phospho- -synuclein by PP2A is methylationdependent.
� In neurodegenerative diseases, inhibition of PP2A demethylation may
offer a novel therapeutic approach towards reducing phosphorylation
proteins such as -synuclein.
This work was supported by funding from the American ����������� Disease Foundation.
*
*
pS129 -syn
pS129 -syn
pS129 -syn
Total -syn
pS129 -syn
pS129 -syn
pS129 -syn
pS129 -syn
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Total -syn
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