alpha -Synuclein Is Phosphorylated by Members of the Src Family of Protein-tyrosine Kinases*

Christopher E. Ellis, Pamela L. Schwartzberg, Teresa L. Grider, Donald W. FinkDagger , and Robert L. Nussbaum§

From the Genetic Diseases Research Branch, National Human Genome Research Institute, National Institutes of Health and the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -Synuclein (alpha -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. alpha -Syn is phosphorylated on serine residues; however, tyrosine phosphorylation of alpha -Syn has not been established (1, 2). A comparison of the protein sequence between Synuclein family members revealed that all four tyrosine residues of alpha -Syn are conserved in all orthologs and beta -Syn paralogs described to date, suggesting that these residues may be of functional importance (3). For this reason, experiments were performed to determine whether alpha -Syn could be phosphorylated on tyrosine residue(s) in human cells. Indeed, alpha -Syn is phosphorylated within 2 min of pervanadate treatment in alpha -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 alpha -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 alpha -Syn can be a target for phosphorylation by the Src family of PTKs.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An ~35-amino acid fragment of alpha -Syn1 was identified initially as a component of amyloid plaques isolated from Alzheimer's diseased brains. The alpha -Syn gene was cloned and found to code for a protein containing 140 amino acids (5). Subsequently, mutations in alpha -Syn have been identified in families with autosomal dominant Parkinson's Disease (6, 7). Additionally, in patients with sporadic PD, alpha -Syn immunoreactivity is detected in Lewy bodies, the pathological hallmark of PD (8). Although, alpha -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 alpha -Syn transgene recapitulate some characteristics of PD suggests that alpha -Syn could be involved directly in the development of this disease (9, 10).

Among the three members of the Syn gene family (alpha , beta , gamma ) alpha -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). alpha -Syn is a small acidic protein containing three discernable regions: an amino-terminal amphipathic repeat region, which can form alpha -helices; a hydrophobic center region found in amyloid plaques; and an acidic carboxyl-terminal region. In solution, alpha -Syn takes on a natively unfolded confirmation; however, in the presence of small vesicles composed of acidic phospholipids it forms an alpha -helical structure. This structure is consistent with its observed binding to synaptic vesicles in vivo (13, 14).

Protein-tyrosine phosphorylation is thought to be important in regulating synaptic function and plasticity (15, 16). Although alpha -Syn was shown recently to be phosphorylated on serine, it has not been determined whether alpha -Syn is also phosphorylated on tyrosine residues (1, 2). The possibility that alpha -Syn might be phosphorylated on tyrosine(s) was initially hypothesized primarily because of the conservation of the four tyrosine residues among alpha -Syn orthologs (3). In addition to the conserved nature of the tyrosine residues in alpha -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 alpha -Syn is phosphorylated on tyrosine.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -Syn were obtained from Dr. Virginia Lee (University of Pennsylvania, Philadelphia, PA) and cultured as above. PP2 was obtained from Calbiochem.

Construction of alpha -Syn Expression Vectors-- alpha -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 alpha -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).

Purification of alpha -Syn from Baculovirus-- Human alpha -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 alpha -Syn was eluted with M imidazole. alpha -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.

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 -80 °C. Protein was quantified using the BCA protein assay kit (Pierce) using bovine serum albumin as a standard.

FLAG-alpha -Syn was isolated from cell lysates by immunoprecipitation using an anti-FLAG M2 affinity resin (Sigma), whereas untagged alpha -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 beta -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.

Metabolic Labeling and Phosphoamino Acid Analysis-- HEK293T cells transiently transfected with FLAG-alpha -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-alpha -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).

In Vitro Kinase Assay-- Kinase assays were performed using 250 ng of denatured rabbit muscle enolase (Sigma) or 500 ng of baculovirus purified His-alpha -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 [gamma -32P]ATP (Amersham Pharmacia Biotech) at 25 °C for 7 min. Kinase reactions were terminated by addition of SDS loading buffer with beta -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

To determine whether alpha -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 alpha -Syn from either pervanadate-treated transiently transfected HEK293T cells (Fig. 1A) or stably transfected HEK293 cells (Fig. 1C) expressing human alpha -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 alpha -Syn (Fig. 1, A and C).3 As illustrated in Fig. 1, A and C, alpha -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 alpha -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, alpha -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).



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Fig. 1.   alpha -Syn is phosphorylated on tyrosine in pervanadate-treated human cells. HEK293T cells transiently transfected with FLAG-alpha -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 alpha -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-alpha -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 alpha -Syn were treated with pervanadate and harvested as described for A except that the alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -Syn in pervanadate-treated HEK293T cells occurs on tyrosine 125. 



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Fig. 2.   Partial sequence of the human alpha -Syn protein illustrating the mutant constructs. All mutants were constructed by site-directed mutagenesis, according to manufacturer's instructions (Stratagene) using the full-length wild-type cDNA as a template. Only amino acid sequences from 25 to 55, 85 to 90, and 120 to 140 are displayed. All constructs contain 140 amino acids except the Y125stop and Y39F/Y125stop constructs, which have only 124 amino acids. The solid lines are indicative of identical amino acids, whereas amino acid substitutions are indicated with single letter designations (X, stop codon). Tyrosine residues are highlighted in bold.



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Fig. 3.   alpha -Syn is phosphorylated primarily on tyrosine 125 in pervanadate-treated human HEK293T cells. A, HEK293T cells were transiently transfected with various FLAG-alpha -Syn constructs or empty vector (control lane) and treated with pervanadate for 20 min. Cells were harvested, alpha -Syn isolated, and Western analysis was performed as described in the legend to Fig. 1 except that instead of using duplicate membranes for normalization, the membranes were consecutively stripped and reprobed with 4G10 and Synuclein-1 antibodies respectively, following immunoblotting with the PY20 antibody. B, values for each mutant construct were semi-quantitatively determined by normalizing the optical density values obtained by laser densitometry from immunoblots using PY20 antibody compared with values obtained using Synuclein-1 antibody. The mean values for each mutant construct, determined from multiple film exposures and from at least three separate experiments, are plotted as a percentage of the value obtained for the wild-type construct (± S.E., B).

To determine the effects of the PD mutations on the tyrosine phosphorylation of alpha -Syn, mutant constructs were transfected into HEK293T cells and compared with the wild-type construct. Tyrosine phosphorylation of wild-type alpha -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 alpha -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 alpha -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 alpha -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 alpha -Syn expressed in HEK293T cells (4).



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Fig. 4.   The Src family kinase inhibitor, PP2, inhibits the pervanadate-induced tyrosine phosphorylation of alpha -Syn in HEK293T cells. HEK293T cells were transiently transfected with FLAG-alpha -Syn, and pretreated with various concentrations (0-50 µM) of PP2 for 1 h followed by pervanadate treatment for 20 min. Cells were harvested and alpha -Syn isolated, and Western analysis was performed using PY20 and Synuclein-1 antibodies, as described in the legend to Fig. 3 (inset). The mean values for each PP2 treatment group were calculated as in Fig. 3 and plotted as a percentage of the value obtained for the vehicle-treated cells (± S.E.).

To determine more directly whether Src family members can phosphorylate alpha -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 alpha -Syn constructs (Fig. 5B) were cotransfected into HEK293T cells along with the PTK expression constructs. Western analysis was then performed on immunoprecipitated alpha -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-alpha -Syn results in increased tyrosine phosphorylation of alpha -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 alpha -Syn under these conditions, indicating that not all cotransfected PTKs are capable of phosphorylating alpha -Syn.



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Fig. 5.   The Src family kinases, c-Src and Fyn, can phosphorylate alpha -Syn in both cotransfection experiments and in in vitro kinase assays. HEK293T cells were cotransfected with various FLAG-alpha -Syn constructs with or without PTK expression vector constructs. Cells were harvested in radioimmune precipitation buffer (A) or CHAPS buffer (B), and alpha -Syn was isolated by immunoprecipitation, as described in the legend to Fig. 1. Immunoprecipitated alpha -Syn was separated in duplicate by SDS-PAGE using 10-20% gradient gels (A, panels 1 and 2; B), whereas total lysates were separated using an 8% gel (A, panels 3 and 4). Western analysis was performed on all samples using PY20, FLAG, SRC2, or GFP antibodies (A and B). Note: Src expression is observed in cells transfected with alpha -Syn without PTK constructs upon longer exposure. Enolase and alpha -Syn were incubated with p59fyn (C, panel 1) or p60c-src (C, panel 2) in kinase buffer containing [gamma -32P]ATP at 25 °C for 7 min. The kinase reactions were terminated by addition of SDS loading buffer w/beta -mercaptoethanol, boiled for 5 min, and separated by SDS-PAGE (10-20%). The gels were dried and exposed to film for 30 s for panel 1 and 10 min for panel 2 to visualize all bands.

As observed in Fig. 3 with pervanadate, c-Src cotransfected with the Y125F alpha -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 alpha -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 alpha -Syn were performed. alpha -Syn (250 or 500 ng) was incubated with 0.4 or 2 units of p59fyn or p60c-src in kinase buffer containing [gamma -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 alpha -Syn are shown in Fig. 5C. alpha -Syn was phosphorylated in vitro by both Fyn (panel 1) and c-Src (panel 2), whereas no phosphorylation was observed in experiments where alpha -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 alpha -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 alpha -Syn, the ratio of the alpha -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 alpha -Syn ~120 times better than c-Src as determined by laser densitometry of multiple exposures.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is of great interest to determine the specific function(s) of alpha -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. alpha -Syn is phosphorylated on serine residues; however, tyrosine phosphorylation of alpha -Syn had not been established (1, 2). A protein alignment of alpha -, beta -, and gamma -Syns revealed that all four of the tyrosine residues of alpha -Syn, located at positions 39, 125, 133, and 136, are conserved in all identified orthologs and beta -Syn paralogs, whereas only the tyrosine at position 39 is conserved in gamma -Syn, suggesting that these residues may be functionally important (3).

The data presented in this manuscript indicate that alpha -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 alpha -Syn directly in cotransfection experiments and in in vitro kinase assays using purified kinases. The data indicate that alpha -Syn can be phosphorylated by the Src family of PTKs and suggest that alpha -Syn is a possible substrate for Src family members in the brain; although, additional experiments are necessary to determine the PTK(s) that phosphorylate alpha -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 alpha -Syn, because its normal function has not been elucidated definitively. alpha -Syn is primarily a soluble protein expressed in presynaptic neurons, but is also loosely associated with synaptic vesicles (29). alpha -Syn is also implicated in regulating a form of dopamine plasticity in an alpha -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 alpha -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 alpha -Syn is intriguing because of overlap in brain region specific expression and subcellular localization between c-Src and alpha -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 alpha -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 alpha -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 alpha -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 alpha -Syn, but not enolase, and therefore is specific for alpha -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 alpha -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 alpha -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 alpha -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 alpha -Syn (41). Colocalization of Tau and alpha -Syn was also demonstrated in axons. Tau associates with the C-terminal region (residues 89-140) of alpha -Syn, through its microtubule binding domain. Jensen and co-workers (41) hypothesized that this interaction between alpha -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 alpha -Syn, thereby enhancing the activity of these kinases for alpha -Syn. It is also conceivable that the association of alpha -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 alpha -Syn to bind and release Tau in a phosphorylation-dependent manner, and thereby contribute to the maintenance of the distal pool of synaptic vesicles.


    ACKNOWLEDGEMENTS

We thank Drs. Virginia Lee and Susan Reuter for the HEK293 cells stably transfected with human alpha -Syn and Drs. Melanie Hartsough, Nelson Cole, and Michael Czar for helpful discussions.


    FOOTNOTES

* 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.


    ABBREVIATIONS

The abbreviations used are: alpha -Syn, alpha -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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