From the Genetic Diseases Research Branch, National Human Genome
Research Institute, National Institutes of Health and the
Laboratory of Stem Cell Biology/Neurotrophic Factors,
Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, Maryland 20892
Received for publication, November 13, 2000
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ABSTRACT |
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An ~35-amino acid fragment of
Among the three members of the Syn gene family ( Protein-tyrosine phosphorylation is thought to be important in
regulating synaptic function and plasticity (15, 16). Although Cell Culture and Transfections--
Human embryonic kidney cells
(HEK293T, gift of Dr. David Baltimore, California Institute of
Technology, Pasadena, CA) were cultured in DMEM containing 10% fetal
bovine serum, 2 mM L-glutamine, 5 mM HEPES, 10 units/ml penicillin, and 10 µg/ml
streptomycin. Cells were transfected using CaPO4
(Stratagene) with Construction of Purification of Western Analysis and Immunoprecipitations--
Transiently
transfected HEK293T cells and stably transfected HEK293 cells were
harvested in CHAPS lysis buffer (0.5% CHAPS, 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 2 mM EGTA, 0.3 mM sodium orthovanadate, and 1×
Protease mixture) or RIPA buffer (1% Triton X-100, 1% deoxycholate,
0.1% SDS, 50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EGTA, and 0.3 mM sodium orthovanadate), snap frozen in a dry ice/ethanol bath and stored at
FLAG- Metabolic Labeling and Phosphoamino Acid Analysis--
HEK293T
cells transiently transfected with FLAG- In Vitro Kinase Assay--
Kinase assays were performed using
250 ng of denatured rabbit muscle enolase (Sigma) or 500 ng of
baculovirus purified His- To determine whether -Synuclein (
-Syn) is implicated in
the pathogenesis of Parkinson's Disease, genetically through missense
mutations linked to early onset disease and pathologically through its
presence in Lewy bodies.
-Syn is phosphorylated on serine residues;
however, tyrosine phosphorylation of
-Syn has not been established
(1, 2). A comparison of the protein sequence between Synuclein family
members revealed that all four tyrosine residues of
-Syn are
conserved in all orthologs and
-Syn paralogs described to date,
suggesting that these residues may be of functional importance (3). For
this reason, experiments were performed to determine whether
-Syn
could be phosphorylated on tyrosine residue(s) in human cells. Indeed,
-Syn is phosphorylated within 2 min of pervanadate treatment in
-Syn-transfected cells. Tyrosine phosphorylation occurs primarily on
tyrosine 125 and was inhibited by PP2, a selective inhibitor of Src
protein-tyrosine kinase (PTK) family members at concentrations
consistent with inhibition of Src function (4). Finally, we demonstrate
that
-Syn can be phosphorylated directly both in cotransfection
experiments using c-Src and Fyn expression vectors and in in
vitro kinase assays with purified kinases. These data
suggest that
-Syn can be a target for phosphorylation by the Src
family of PTKs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Syn1 was identified
initially as a component of amyloid plaques isolated from Alzheimer's
diseased brains. The
-Syn gene was cloned and
found to code for a protein containing 140 amino acids (5).
Subsequently, mutations in
-Syn have been identified in
families with autosomal dominant Parkinson's Disease (6, 7).
Additionally, in patients with sporadic PD,
-Syn
immunoreactivity is detected in Lewy bodies, the pathological hallmark
of PD (8). Although,
-Syn is linked to the two most
common neurodegenerative disorders, its role in the pathogenesis of
these diseases is unknown. The recent observation that both mice and
flies expressing a human
-Syn transgene recapitulate some
characteristics of PD suggests that
-Syn could be involved directly
in the development of this disease (9, 10).
,
,
)
-Syn is the best characterized. It is most highly
expressed in presynaptic neurons of the brain with greater abundance in
areas of the hippocampus and cortex (3, 11, 12).
-Syn is a small
acidic protein containing three discernable regions: an amino-terminal
amphipathic repeat region, which can form
-helices; a hydrophobic
center region found in amyloid plaques; and an acidic carboxyl-terminal region. In solution,
-Syn takes on a natively unfolded confirmation; however, in the presence of small vesicles composed of acidic phospholipids it forms an
-helical structure. This structure is
consistent with its observed binding to synaptic vesicles in vivo (13, 14).
-Syn
was shown recently to be phosphorylated on serine, it has not been
determined whether
-Syn is also phosphorylated on tyrosine residues
(1, 2). The possibility that
-Syn might be phosphorylated on
tyrosine(s) was initially hypothesized primarily because of the
conservation of the four tyrosine residues among
-Syn orthologs (3).
In addition to the conserved nature of the tyrosine residues in
-Syn, at least one of these residues contains flanking sequences that share homology with established tyrosine phosphorylation sites.
Given the importance of tyrosine phosphorylation in the regulation of
many cellular processes, we examined whether
-Syn is phosphorylated
on tyrosine.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Syn, FynT, Rlk-GFP, and wild-type and mutant
c-Src (K295M) expression vectors (17, 18). HEK293 cells (ATCC CRL-1573)
stably transfected with human
-Syn were obtained from Dr. Virginia
Lee (University of Pennsylvania, Philadelphia, PA) and cultured as
above. PP2 was obtained from Calbiochem.
-Syn Expression Vectors--
-Syn was
isolated by reverse transcriptase-PCR using human lymphoblastoid cell
DNA.2 PCR mutagenesis was
performed using the QuickChange site-directed mutagenesis kit
(Stratagene). Amino-terminal FLAG-tagged
-Syn was constructed by PCR
using the primer sequences
GCTCTAGAGCCACCATGGATTACAAGGATGACGACGATAAGGATGTATTCA (5') and
CCGCTCGAGGGCTTCAGGTTCGTAGTCTTGATA (3') (Life Technologies, Inc.) by
standard methods, and was ligated into pcDNA 3.1 vector (Invitrogen). All constructs were sequenced on both strands by Seqwright (Houston, TX).
-Syn from Baculovirus--
Human
-Syn was
subcloned into pBlueBacHis vector, and virus production and expression
in SF9 cells were performed as described (Invitrogen). The cell pellet
was resuspended in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM imidazole, and 1× protease
mixture (Sigma), and the lysate was boiled for 10 min. The cleared
lysate was bound to Talon resin (CLONTECH), and
-Syn was eluted with 1 M imidazole.
-Syn-containing
fractions were identified by SDS-PAGE, pooled, dialyzed against 20 mM Tris-HCl, pH 7.5 and 100 mM NaCl and stored
at
80 °C.
80 °C. Protein was
quantified using the BCA protein assay kit (Pierce) using bovine serum
albumin as a standard.
-Syn was isolated from cell lysates by immunoprecipitation
using an anti-FLAG M2 affinity resin (Sigma), whereas untagged
-Syn
was isolated using a monoclonal antibody (202) cross-linked to protein
A-agarose beads (Roche Molecular Biochemicals) using dimethylpimelimidate (ICN) as the cross-linking agent (19, 20). Immunoprecipitants were boiled 5 min in SDS loading buffer (50 mM Tris-HCL, pH 6.8, 2% SDS, 10% glycerol, and 0.1%
bromphenol blue). Total lysates were boiled in SDS loading buffer
containing
-mercaptoethanol. Proteins were separated by
SDS-PAGE (Tris-Glycine, Novex), transferred to Hybond-P membrane
(Amersham Pharmacia Biotech), immunoblotted using PY20 (Transduction
Laboratories) (1:1500), Synuclein-1 (Transduction Laboratories)
(1:5000), 4G10 (Upstate Biotechnology Inc.) (1:3000), GFP (Roche)
(1:1000), FLAG (Sigma) (1:1500), and SRC2 antibodies (Santa Cruz
Biotechnology) (1:500) and detected by enhanced chemiluminesence (ECL,
Amersham Pharmacia Biotech). Membranes were blocked in 1% bovine serum
albumin/Tris-buffered saline, 0.1% Tween 20 for PY20, 4G10, and SRC2
immunoblots, and 5% nonfat milk/phosphate-buffered saline, 0.1% Tween
20 for GFP, Synuclein-1, and FLAG immunoblots. Laser densitometry
(Molecular Dynamics) was performed on multiple exposures from at least
three experiments and analyzed using NIH Image software.
-Syn were labeled with
H332PO4 (ICN) in phosphate-free
DMEM medium for 4 h. Following immunoprecipitation, samples were
separated by SDS-PAGE, transferred to Hybond-P membrane (Amersham
Pharmacia Biotech), and autoradiography was performed. 32P-labeled FLAG-
-Syn was excised from the membrane,
hydrolyzed in HCL, and phosphoamino acids were resolved by
two-dimensional electrophoresis as described previously (21). Unlabeled
phosphoamino acid standards were visualized by ninhydrin staining, and
radiolabeled phosphoamino acids were detected using a phosphorimager
(Molecular Dynamics, model 425E).
-Syn as substrates (22). Substrates were
incubated with 0.5 or 2 units of p59fyn or
p60c-src (Upstate Biotechnology) in 30 µl of
kinase buffer (20 mM Tris-HCL, pH 7.4 and 5 mM
MnCl2 containing 10 µCi [
-32P]ATP
(Amersham Pharmacia Biotech) at 25 °C for 7 min. Kinase reactions
were terminated by addition of SDS loading buffer with
-mercaptoethanol, boiled for 5 min, separated by SDS-PAGE
(10-20%), and exposed to film. According to the manufacturer, the
c-Src and Fyn preparations do not contain contaminating kinases.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Syn can be phosphorylated on tyrosine in
a human cell line, the protein-tyrosine phosphatase (PTP) inhibitor
pervanadate was utilized. Pervanadate, a complex of vanadate and
hydrogen peroxide, is a competitive inhibitor of PTPs that works by
irreversible oxidation and functions on intact cells because of its
cell permeability (23). Immunoprecipitated
-Syn from either
pervanadate-treated transiently transfected HEK293T cells (Fig.
1A) or stably transfected
HEK293 cells (Fig. 1C) expressing human
-Syn was
separated by SDS-PAGE in duplicate. Western analysis was then performed
with either PY20, a phosphotyrosine specific antibody, or Synuclein-1
antibody, which specifically recognizes
-Syn (Fig. 1, A
and C).3 As
illustrated in Fig. 1, A and C,
-Syn is
phosphorylated on tyrosine residues within 2 min of pervanadate
treatment and increases incrementally over 20 min. Cells treated with
sodium orthovanadate or hydrogen peroxide alone resulted in no change
in
-Syn phosphorylation (data not shown). Tyrosine phosphorylation
was confirmed by phosphoamino acid analysis (Fig. 1B).
Phosphorylated serine but not threonine residues were also observed,
confirming the results reported previously (1). Thus,
-Syn is
phosphorylated on tyrosine in both a time- and
dose-dependent manner following inhibition of PTPs by
pervanadate (Fig. 1, A and C, data not
shown).
View larger version (42K):
[in a new window]
Fig. 1.
-Syn is phosphorylated on
tyrosine in pervanadate-treated human cells. HEK293T cells
transiently transfected with FLAG-
-Syn were treated for different
times (0-20 min) with pervanadate (100 µM sodium
orthovanadate and 4 mM H2O2). Cells
were harvested in CHAPS lysis buffer, and
-Syn was isolated by
immunoprecipitation using an anti-FLAG affinity resin. Resulting
immunoprecipitants were separated in duplicate by SDS-PAGE (10-20%
Tris-glycine) and transferred to PVDF membrane. A,
immunoblots were performed on duplicate membranes using PY20 or
Synuclein-1 antibodies and visualized by ECL. B, for
phosphoamino acid analysis, transiently transfected cells were labeled
with [32P]H3PO4. FLAG-
-Syn was
isolated from cell lysates as stated above, and phosphoamino acid
analysis was performed as outlined under "Materials and Methods."
The positions of phosphorylated serine (pS), threonine
(pT), and tyrosine (pY) standards are
illustrated. C, HEK293 cells stably expressing human
-Syn
were treated with pervanadate and harvested as described for
A except that the
-Syn (202) antibody was used for
immunoprecipitation.
To map the phosphorylated tyrosine residue(s), mutants were constructed
by exchanging one or more of four tyrosine residues in -Syn with
either a phenylalanine or a stop codon (Fig.
2). These mutant constructs were
transfected into HEK293T cells and treated for 20 min with pervanadate.
Western analysis performed on immunoprecipitated
-Syn, using PY20
and 4G10 phosphotyrosine specific antibodies or Synuclein-1 antibody,
indicates that the Y125F mutant construct is the only single tyrosine
mutation that results in a significant effect, reducing tyrosine
phosphorylation to ~5% of the wild-type control (Fig.
3). Tyrosine phosphorylation of the other
single tyrosine mutation constructs was not significantly different
from the wild-type construct. Phosphorylation of the Y133F mutant
construct appeared to be greater than the wild-type; however, this
difference was not statistically significant (Student's t
test). Interestingly, the Y133/136F double mutation resulted in an
~75% reduction in the tyrosine phosphorylation of
-Syn. Because
neither single mutation alone results in a significant reduction in
tyrosine phosphorylation, a possible explanation for this decrease is
that this double mutation disrupts the interaction of
-Syn with a
protein involved in phosphorylating tyrosine 125. Longer exposures
reveal a very low level of phosphorylation of tyrosines 39, 133, and
136, but no phosphorylation in untransfected control cells or in
Y39F/Y125stop-transfected cells, which do not contain any tyrosine
residues. These data suggest that the majority of tyrosine
phosphorylation of
-Syn in pervanadate-treated HEK293T cells occurs
on tyrosine 125.
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To determine the effects of the PD mutations on the tyrosine
phosphorylation of -Syn, mutant constructs were transfected into
HEK293T cells and compared with the wild-type construct. Tyrosine
phosphorylation of wild-type
-Syn in pervanadate-treated cells does
not differ significantly from that of cells transfected with PD
mutation constructs (A30P and A53T, data not shown). Additionally, the
effect of mutating serines at positions 87 and 129 to alanine on
tyrosine phosphorylation was determined, because these serines were
shown previously to be phosphorylated (1). Phosphorylation of serines
87 and 129 is not necessary for phosphorylation of tyrosine 125 under
the conditions tested (data not shown).
To help elucidate the PTK(s) that may be involved in the tyrosine
phosphorylation observed with pervanadate, PP2, a selective inhibitor
of the Src family of PTKs, was incubated with the cells as indicated in
Fig. 4 (4). Western analysis revealed
that these cells do express Src family members (data not shown).
Wild-type -Syn was transfected into HEK293T cells and pretreated
with various concentrations of PP2 or a Me2SO-vehicle
control for 1 h prior to treatment with pervanadate. Western
analysis was then performed on immunoprecipitated
-Syn using the
PY20 and Synuclein-1 antibodies, and mean values for each PP2 treatment
group were plotted as a percentage of the vehicle-treated control (Fig.
4). PP2 inhibits the pervanadate-induced tyrosine phosphorylation of
-Syn with an EC50 of ~1 µM, a
concentration reported to inhibit effectively the function of Src
family PTKs in human T-cells, suggesting that Src family PTKs are able
to phosphorylate
-Syn expressed in HEK293T cells (4).
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To determine more directly whether Src family members can phosphorylate
-Syn in these cells, cotransfection experiments were performed
utilizing c-Src and Fyn expression vectors as depicted in Fig.
5A. Additionally, a mutant
c-Src (K295M), which inactivates the kinase by disrupting its
phosphotransfer activity, and Rlk, a nonreceptor PTK whose expression
is limited to T-cells and mast cells, were utilized as controls
(24-26). Wild-type (Fig. 5A) and various mutant
-Syn
constructs (Fig. 5B) were cotransfected into HEK293T cells
along with the PTK expression constructs. Western analysis was then
performed on immunoprecipitated
-Syn using the PY20 and FLAG
antibodies (Fig. 5, A, panels 1 and
2; B), or on total lysates using SRC2 and GFP
antibodies (Fig. 5A, panels 3 and 4).
As illustrated in Fig. 5A, c-Src and Fyn, but not the mutant
c-Src (K295M) or Rlk·GFP, cotransfected with FLAG-
-Syn results in
increased tyrosine phosphorylation of
-Syn. Western analysis of
total lysates confirmed not only that c-Src, Fyn, and Rlk·GFP are
expressed, but also that these PTKs are active in these cells, because
large increases in tyrosine phosphorylation is observed in cells
transfected with these PTKs compared with untransfected and mutant
c-Src (K295M)-transfected cells (data not shown). Although Rlk is
active in these cells, it does not phosphorylate
-Syn under these
conditions, indicating that not all cotransfected PTKs are capable of
phosphorylating
-Syn.
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As observed in Fig. 3 with pervanadate, c-Src cotransfected with the
Y125F -Syn mutant construct also results in a significant reduction
in tyrosine phosphorylation as compared with the wild-type construct,
whereas other single amino acid changes do not differ significantly
(Fig. 5B). This indicates that tyrosine 125 is also the
major tyrosine phosphorylation site in c-Src-cotransfected cells. Src
family PTKs could be phosphorylating
-Syn directly, or indirectly by
acting through Src-activated pathways involving other kinase(s). To
help distinguish between these two possibilities, in vitro
kinase assays using purified c-Src, Fyn, and
-Syn were performed.
-Syn (250 or 500 ng) was incubated with 0.4 or 2 units of
p59fyn or p60c-src in
kinase buffer containing [
-32P]ATP. Enolase (250 ng)
was used as a positive control, because it is a substrate for c-Src and
Fyn (22, 27). The reactions were separated by SDS-PAGE, and
autoradiography was performed. Results using 2 units of kinase and 500 ng of
-Syn are shown in Fig. 5C.
-Syn was
phosphorylated in vitro by both Fyn (panel 1) and
c-Src (panel 2), whereas no phosphorylation was observed in
experiments where
-Syn was incubated without PTKs or if bovine serum
albumin was provided as a substrate (data not shown). Fyn and c-Src
could also phosphorylate
-Syn at lower concentrations of kinase (0.4 units) and substrate (250 ng) than those shown in Fig. 5C
(data not shown).
To compare the relative ability of Fyn and c-Src to phosphorylate
-Syn, the ratio of the
-Syn signal versus the enolase signal for each kinase was compared. The ability of Fyn and c-Src to
phosphorylate enolase under the conditions tested was approximately the
same per unit of kinase, although not readily apparent because different exposure times are shown in panels 1 and
2 in Fig. 5C. Relative to enolase, Fyn
phosphorylated
-Syn ~120 times better than c-Src as determined by
laser densitometry of multiple exposures.
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DISCUSSION |
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It is of great interest to determine the specific function(s) of
-Syn because of its potential importance in the
pathogenesis of PD. Studying post-translational modifications, such as
phosphorylation, can be very useful in gaining insight into protein
function.
-Syn is phosphorylated on serine residues; however,
tyrosine phosphorylation of
-Syn had not been established (1, 2). A
protein alignment of
-,
-, and
-Syns revealed that all four of
the tyrosine residues of
-Syn, located at positions 39, 125, 133, and 136, are conserved in all identified orthologs and
-Syn
paralogs, whereas only the tyrosine at position 39 is conserved in
-Syn, suggesting that these residues may be functionally important
(3).
The data presented in this manuscript indicate that -Syn is
phosphorylated on tyrosine in response to pervanadate inhibition of
PTPs. This tyrosine phosphorylation occurs primarily on tyrosine 125 and is inhibited by PP2, implicating the involvement of the Src family
of PTKs. Additionally, specific members of the Src family of PTKs,
c-Src and Fyn, phosphorylate
-Syn directly in cotransfection
experiments and in in vitro kinase assays using purified
kinases. The data indicate that
-Syn can be phosphorylated by the
Src family of PTKs and suggest that
-Syn is a possible substrate for
Src family members in the brain; although, additional experiments are
necessary to determine the PTK(s) that phosphorylate
-Syn in
vivo.
Sequences flanking tyrosine 125, as depicted in Fig. 2, closely
resemble those of the optimal substrate sequences for PTKs determined
using an oriented peptide library technique (28). For example, this
tyrosine 125 site (DNEAYEMP) is similar to the c-Src
optimal substrate sequence which was determined to be
DEEIY(G/E)EF. The amino acids flanking tyrosine 125 at
positions 4,
2, and +1 are identical to this optimal substrate
sequence, whereas the alanine at position
1 is similar to the
isoleucine in the ideal sequence in that they are both hydrophobic
residues. Although the methionine and proline residues at positions +2
and +3, relative to the phosphorylated tyrosine, are not optimal
substrate residues for c-Src phosphorylation, they are optimal residues
for phosphorylation by other PTKs (28). Thus, the tyrosine 125 site is
consistent with optimal substrate sequences of PTKs including
c-Src.
It is difficult to speculate on the functional consequences of tyrosine
phosphorylation of -Syn, because its normal function has not been
elucidated definitively.
-Syn is primarily a soluble protein
expressed in presynaptic neurons, but is also loosely associated with
synaptic vesicles (29).
-Syn is also implicated in regulating a form
of dopamine plasticity in an
-Syn knockout mouse model,
and maintenance of the distal pool of synaptic vesicles in primary
hippocampal cultures (30, 31). Covalent modification, such as
phosphorylation, is a likely candidate for regulation of
-Syn at the
synapse and could be important in modulating its function. Tyrosine
phosphorylation occurs in synaptic vesicles and is important for
regulating synaptic function in the brain (15, 32). For example,
tyrosine kinase inhibitors have been shown to block long-term
potentiation in the hippocampus, and increased tyrosine phosphorylation
in the squid giant synapse modulates synaptic transmission by
increasing calcium currents, both implicating PTKs in synaptic
plasticity (16, 33). Fyn and c-Src are also thought to be involved in
spatial learning and synaptic plasticity (34-36). For example, Fyn
knockout mice exhibit an impairment in memory and learning, which is
thought to be caused by alterations in long-term potentiation (35).
The Src family of nonreceptor PTKs is expressed ubiquitously with
increased expression in neurons and hematopoietic cells (37). The
possibility that members of the Src family of PTK(s) may be involved in
the modification of -Syn is intriguing because of overlap in brain
region specific expression and subcellular localization between c-Src
and
-Syn. Both proteins are expressed highly in the brain and also
have overlapping expression in various regions, for example, in the
hippocampus (3, 11, 36). Subcellularly, both of these proteins are
loosely associated with synaptic vesicles in presynaptic neurons (12,
38). c-Src has also been shown to be active at the synapse by
phosphorylating synaptic vesicle proteins such as synaptophysin and
synaptogyrin (38).
The apparent 120-fold-increased phosphorylation of -Syn by Fyn
versus c-Src, relative to enolase, raises two interesting possibilities that could account for this increase (Fig.
5C). The first is that
-Syn is a more preferred substrate
for Fyn than for c-Src. The second is that some enhancing factor is
copurifying with Fyn because it was purified by sequential
chromatography from a membrane fraction of bovine thymus, whereas
recombinant human c-Src was purified from baculovirus. We believe that
the latter explanation is more likely because no significant difference in the relative amount of tyrosine phosphorylation of
-Syn exists in
cotransfection experiments comparing c-Src and Fyn despite similar
protein expression (Fig. 5A, panels 1-3). This
hypothesized factor enhances greatly the in vitro activity
of Fyn for
-Syn, but not enolase, and therefore is specific for
-Syn. To prove this hypothesis Fyn and c-Src must be isolated by an
equivalent means, allowing for direct comparison and ultimately one
must isolate and identify this "enhancing factor".
Although the functional consequences of phosphorylation of the tyrosine
125 residue of -Syn remain to be elucidated, it could regulate its
ability to bind synaptic vesicles and/or be important in regulating
protein/protein interactions. It has been reported that phosphorylation
of the serine 129 residue of
-Syn results in a reduction in binding
to phospholipid containing liposomes (2). Additionally, phosphorylation
regulates the association of the synaptic vesicle protein, Synapsin I
to synaptic vesicles (39). Similarly, tyrosine phosphorylation may
allow for the dynamic regulation of
-Syn binding to synaptic
vesicles. Interestingly, Synapsin I has also been shown to interact
with c-Src and reportedly increases c-Src tyrosine kinase activity
(40).
Recently, the microtubule-associated protein Tau was identified as a
binding partner of -Syn (41). Colocalization of Tau and
-Syn was
also demonstrated in axons. Tau associates with the C-terminal region
(residues 89-140) of
-Syn, through its microtubule binding domain.
Jensen and co-workers (41) hypothesized that this interaction
between
-Syn and Tau could link synaptic vesicles with microtubules.
Tau has also been shown to both colocalize and interact directly with
the Src PTK family member, Fyn (42). Hypothetically, Tau could bring
Src PTK family members, such as Fyn, into close proximity to
-Syn,
thereby enhancing the activity of these kinases for
-Syn. It is also
conceivable that the association of
-Syn and Tau could be regulated
by phosphorylation of tyrosine 125, and that in certain populations of
synaptic vesicles could regulate their attachment to microtubules. This
could allow
-Syn to bind and release Tau in a
phosphorylation-dependent manner, and thereby contribute to
the maintenance of the distal pool of synaptic vesicles.
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ACKNOWLEDGEMENTS |
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We thank Drs. Virginia Lee and Susan Reuter
for the HEK293 cells stably transfected with human -Syn and Drs.
Melanie Hartsough, Nelson Cole, and Michael Czar for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Genetic Diseases Research Branch, NHGRI, National Institutes of Health, 49 Convent Dr., MSC 4472, Bethesda, MD 20892-4472. Tel.: 301-402-2039; Fax: 301-402-2170; E-mail: rlnuss@nhgri.nih.gov.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M010316200
2 R. L. Nussbaum, unpublished data.
3 C. E. Ellis, D. E. Cobin, and R. L. Nussbaum, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
-Syn,
-synuclein;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
DMEM, Dulbecco's modified Eagle's medium;
PTP, protein-tyrosine phosphatase;
PTK, protein-tyrosine kinase;
PD, Parkinson's disease;
GFP, green fluorescent protein;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
HEK, human embryonic kidney cells.
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