Activation of Pyk2/RAFTK induces tyrosine phosphorylation of
K-synuclein via Src-family kinases
Takeshi Nakamuraa, Hiroshi Yamashitaa;?, Yoshito Naganoa, Tetsuya Takahashia,
Shalom Avrahamb, Hava Avrahamb, Masayasu Matsumotoa, Shigenobu Nakamuraa
aDepartment of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi,
Hiroshima 734-8551, Japan
bDivision of Experimental Medicine, Beth Israel-Deaconess Medical Center, Harvard Institute of Medicine, 4 Blackfan Circle,
Boston, MA 02115, USA
Received 23 April 2002; accepted 28 April 2002
First published online 29 May 2002
Edited by Jesus Avila
implicated in the pathogenesis of Parkinson’s disease. The
present report demonstrates that the protein tyrosine kinase
Pyk2/RAFTK is involved in cell stress-induced tyrosine phos-
phorylation of K KS. Hyperosmotic stress induced tyrosine
phosphorylation of K KS via Pyk2/RAFTK at tyrosine residue
125. Pyk2/RAFTK-mediated phosphorylation of K KS was pri-
marily achieved with Src-family kinases. In addition, osmotic
stress-induced phosphorylation of K KS was dependent on Pyk2/
RAFTK activation. Accordingly, such results indicate that Pyk2/
RAFTK lies upstream of Src-family kinases in the signaling
cascade by which osmotic stress induces tyrosine phosphorylation
of K KS. ? 2002 Published by Elsevier Science B.V. on behalf of
the Federation of European Biochemical Societies.
K K-Synuclein (K KS) is a neuronal protein that has been
Key words: K-Synuclein; Tyrosine phosphorylation;
Pyk2/RAFTK; Osmotic stress; Parkinson’s disease
K-Synuclein (KS) is a 140-residue soluble neuronal protein
of unknown function that has been implicated in the patho-
genesis of Parkinson’s disease. Abnormal aggregates of KS are
the main component of Lewy bodies, which represent a hall-
mark ¢nding of Parkinson’s disease . Molecular genetic
studies have identi¢ed two di¡erent point mutations, A30P
and A53T, in the KS gene that causes familial Parkinson’s
disease [2,3]. In neuronal cells, KS is especially abundant in
presynaptic terminals and is well known to be phosphorylated
by several kinases [4^8]. Recently, another group and our own
group have reported that KS is phosphorylated by members of
the Src-family of protein tyrosine kinases (PTKs) [7,8]. None-
theless, the mechanisms controlling phosphorylation of KS by
Src-family kinases remain poorly understood.
Pyk2/RAFTK is a member of the focal adhesion kinase
(FAK) family of PTKs, alternatively known as CAKL,
FAK-2, or CADTK . Pyk2/RAFTK can be activated not
only by a variety of extracellular signals that elevate the intra-
cellular Ca2þconcentration, which results in protein kinase C
activation, but also by stress signals . In the central nervous
system, Pyk2/RAFTK is present in abundance in synapses
and appears to correlate with long-term potentiation . Ac-
tivation of Pyk2/RAFTK leads to modulation of ion channel
function and activation of a mitogen-activated protein kinase
(MAPK) signaling pathway . Consequently, the action of
Pyk2/RAFTK remains crucial for not only the control of
di¡erentiation and survival of neuronal cells, but also the
regulation of neuronal excitability, plasticity, and memory
. In addition, Pyk2/RAFTK interacts with several proteins
involved in integrin signaling, including paxillin and c-Src.
The Src-binding site, tyrosine 402, represents an autophos-
phorylation site for Pyk2/RAFTK . Following autophos-
phorylation, c-Src is recruited and the complex is able to
phosphorylate several proteins. For example, in PC12 cells,
c-Src binds to tyrosine 402 of Pyk2/RAFTK following stim-
ulation with bradykinin, lysophosphatidic acid, and depolar-
The present study demonstrates that KS is phosphorylated
at a tyrosine residue via activation of Pyk2/RAFTK in re-
sponse to cell stress. Moreover, it was also demonstrated
that cooperation between Pyk2/RAFTK and Src-family ki-
nases is required for KS phosphorylation.
2. Materials and methods
2.1. Antibodies, cells, and expression vectors
Anti-hemagglutinin (HA) and anti-c-Src antibodies were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phos-
photyrosine monoclonal antibody (P-Tyr) was purchased from Cell
Signaling Technology (Beverly, MA, USA). Anti-Pyk2/RAFTK and
anti-KS antibodies were purchased from BD Transduction Laborato-
ries (Lexington, KY, USA). M2 monoclonal antibody against the
FLAG epitope was purchased from Sigma (St. Louis, MO, USA).
COS7 cells and full-length KS cDNA were obtained from the Amer-
ican Tissue Culture Collection. cDNAs encoding wild-type and mu-
tant (A30P, A53T, Y39F, Y125F, Y133F, and Y136F) KS were gen-
erated and subcloned into pcDNA3-HA vectors as previously
described [6,7]. The c-Src expression vector was purchased from Up-
state Biotechnology (Lake Placid, NY, USA). cDNAs encoding wild-
type and mutant (Y402F and K457R) Pyk2/RAFTK were generated
and subcloned into pcDNA3-FLAG vectors as previously described
2.2. Expression of cDNA constructs in COS7 cells; cell stimulation
with osmotic stress
COS7 cells were grown in Dulbecco’s modi¢ed Eagle’s medium
(DMEM) containing 10% fetal bovine serum and antibiotics. Trans-
0014-5793/02/$22.00 ? 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
PII: S0 014-57 93(02)028 61-2
*Corresponding author. Fax: (81)-82-505 0490.
E-mail address: email@example.com (H. Yamashita).
FEBS 26180 FEBS Letters 521 (2002) 190^194
fection of expression vectors into cells was performed by FuGENE 6
(Roche, Indianapolis, IL, USA) according to the manufacturer’s pro-
tocol. Cells were subjected to an assay 24 h post-transfection. Prior to
stimulation, cells were starved for 18 h in DMEM containing 0.1%
fetal bovine serum. Cells were then stimulated with osmotic stress by
exposure to 300 mM D-sorbitol (Sigma) in culture medium at 37‡C for
indicated time periods. Ethylene glycol-bis(2-aminoethyl)-N,N,NP,NP-
tetraacetic acid(EGTA) and
N,N,NP,NP-tetraacetic acid tetrakis (BAPTA-AM) were obtained
from Sigma. PP2 was obtained from Calbiochem (San Diego, CA,
2.3. Immunoprecipitation and immunoblotting
After sorbitol treatment, cells were lysed with lysis bu¡er (50 mM
Tris^HCl (pH 7.4), 10% glycerol, 150 mM NaCl, 1 mM EDTA, 1%
Triton X-100, 1% sodium deoxycholate, 1 mM phenylmethylsulfonyl
£uoride, 1 mM sodium orthovanadate, 25 mM NaF, 10 Wg/ml leu-
peptin, and 10 Wg/ml aprotinin). After the lysate protein contents were
normalized using a protein assay kit (Bio-Rad, Hercules, CA, USA),
the cell lysate (500 Wg/sample) was immunoprecipitated with anti-
FLAG or anti-HA antibodies using protein G-Sepharose beads
(Pierce, Rockford, IL, USA). Each immunoprecipitate was divided
into two parts, separated on SDS^polyacrylamide gel electrophoresis
(SDS^PAGE), transferred onto nitrocellulose membranes (Schlei-
cherpSchuell, Dassel, Germany), and subjected to immunoblotting
with anti-Pyk2/RAFTK, anti-KS, or anti-P-Tyr antibodies. The blots
were developed using the ECL system (NEN, Boston, MA, USA).
The degree of KS phosphorylation was quantitated by densitometric
analysis of non-saturated radiographs with NIH Image software.
3. Results and discussion
3.1. Pyk2/RAFTK and c-Src are involved in osmotic
stress-induced phosphorylation of KS
Other groups and our own group have reported that Src-
family kinases phosphorylate KS [7,8]. Since Pyk2/RAFTK is
highly expressed in the human brain, and is involved in var-
ious signaling pathways, to include cell stress signals in neuro-
nal cells, the present study investigated whether or not hyper-
osmotic stress induced phosphorylation of KS. COS7 cells
were transfected with HA-tagged expression vectors for KS
and FLAG-tagged expression vectors for either PTK (c-Src
or FAK) or Pyk2/RAFTK. After 24 h, the cells were starved
for 18 h, and then subjected to hyperosmotic stress by treat-
ment with 300 mM D-sorbitol for 10 min. Immunoblotting
with anti-P-Tyr antibodies revealed that osmotic stress in-
duced tyrosine phosphorylation of KS in both untransfected
and transfected cells (Fig. 1A). In untransfected COS7 cells,
the tyrosine phosphorylation level of KS was low after sorbitol
treatment, despite endogenous expression of c-Src in COS7
cells (Fig. 3). Expression of c-Src or Pyk2/RAFTK increased
the phosphorylation level of KS from 1.8 to 15.0 or 14.3
(n=4), respectively; expression of FAK, however, had no
e¡ect on the tyrosine phosphorylation level of KS (Fig. 1A).
Fig. 1. Pyk2/RAFTK is involved in phosphorylation of KS at tyrosine residue 125 in response to osmotic stress. A: COS7 cells were cotrans-
fected with HA-tagged expression vectors for KS and FLAG-tagged expression vectors for c-Src, FAK, or Pyk2/RAFTK, as indicated. Starved
cells were treated with 300 mM D-sorbitol (Osm) for 10 min at 37‡C and compared with untreated cells subjected to the same conditions. Each
cell lysate was immunoprecipitated with anti-FLAG or anti-HA antibodies. The immunoprecipitates were then separated on 15% SDS^PAGE
and immunoblotted with anti-FLAG, anti-KS, or anti-phosphotyrosine (P-Tyr). Densitometric analysis of KS phosphorylation is exhibited in
the lower panel. Results are means?S.E.M. for four independent experiments. B: COS7 cells cotransfected with expression vectors for FLAG-
Pyk2/RAFTK and HA-KS (WT, A30P, A53T, Y39F, Y125F, Y133F, and Y136F) were treated with 300 mM D-sorbitol (Osm), lysed, and com-
pared with untreated cells subjected to the same conditions. Each cell lysate was immunoprecipitated with anti-FLAG or anti-HA antibodies
and analyzed by SDS^PAGE and immunoblotting with anti-FLAG, anti-KS, or anti-P-Tyr antibodies. C: Concentration dependence of osmotic
stress-induced phosphorylation of KS. COS7 cells were treated with di¡erent concentrations of D-sorbitol (1, 10, 50, 100, 300, and 600 mM) for
10 min at 37‡C and compared with untreated cells subjected to the same conditions. Cells were prepared and subjected to immunoprecipitation,
immunoblotting, and densitometric analysis of KS phosphorylation. Results are means?S.E.M. for four independent experiments. D: Time
course of osmotic stress-induced phosphorylation of KS. COS7 cells were treated with 300 mM sorbitol for di¡erent periods of time (0, 1, 5,
10, 30 min) and compared with untreated cells. Cells were prepared and subjected to immunoprecipitation, immunoblotting, and densitometric
analysis of KS phosphorylation. Results are means?S.E.M. for four independent experiments.
T. Nakamura et al./FEBS Letters 521 (2002) 190^194
In addition, c-Src and Pyk2/RAFTK, but not FAK, were
phosphorylated and activated in response to osmotic stress.
Such results suggest that both c-Src and Pyk2/RAFTK are
involved in osmotic stress-dependent phosphorylation of KS.
It should also be noted that it was found that COS7 cells do
not endogenously express Pyk2/RAFTK. The above results
suggest that other pathways might exist for KS phosphoryla-
tion via Src-family kinases.
3.2. Characterization of Pyk2/RAFTK-mediated tyrosine
phosphorylation of KS
An additional experiment was performed to determine
whether or not A30P and A53T mutations of KS could a¡ect
the phosphorylation state of KS under conditions of osmotic
stress. COS7 cells expressing FLAG-tagged Pyk2/RAFTK
and HA-tagged KS (wild-type, A30P, or A53T) were treated
with sorbitol, lysed, and immunoprecipitated with anti-FLAG
or anti-HA antibodies. Immunoblotting with anti-P-Tyr anti-
bodies revealed that osmotic stress induced phosphorylation
of not only wild-type KS, but also mutant KS, suggesting that
A30P and A53T mutations do not a¡ect Pyk2/RAFTK-medi-
ated phosphorylation of KS (Fig. 1B). KS contains four tyro-
sine residues: Y39, Y125, Y133, and Y136. To determine the
tyrosine phosphorylation site utilized following osmotic stress,
COS7 cells were transfected with expression vectors for
FLAG-Pyk2/RAFTK and HA-KS with Y^F single substitu-
tions: Y39F, Y125F, Y133F, and Y136F. Immunoblotting
with anti-P-Tyr antibodies revealed that osmotic stress in-
duced phosphorylation of Y39F, Y133F, and Y136F, but
not Y125F, suggesting that the tyrosine residue phosphorylat-
ed was 125 (Fig. 2B). This tyrosine residue has been reported
to be phosphorylated by Src-family kinases [7,8]. c-Src
and Fyn can directly phosphorylate KS, however phos-
phorylation of KS by Pyk2/RAFTK was not observed in
vitro . Although the issue of whether or not Pyk2/RAFTK
directly phosphorylates KS in vivo remains a controversial
topic of discussion, KS was found to be phosphorylated in
response to osmotic stress when coexpressed with Pyk2/
Subsequently, the concentration dependence for osmotic
stress-induced phosphorylation of KS was examined. COS7
cells transfected with expression vectors for FLAG-Pyk2/
RAFTK and HA-KS were treated for 10 min with sorbitol
at various concentrations: 1, 10, 50, 100, 300, and 600 mM.
Treatment with sorbitol induced phosphorylation of KS in a
concentration-dependent manner (Fig. 1C). Densitometric
analysis demonstrated that phosphorylation of KS was max-
imal at V14.8-fold above baseline (n=4) when cells were
treated with 300 mM sorbitol; higher concentrations resulted
in a decline of phosphorylation. Subsequently, the time course
for KS phosphorylation was examined. Cells were stimulated
with sorbitol for di¡erent periods of time: 0, 1, 5, 10, and 30
min. Phosphorylation of KS was rapid, becoming prominent
at 1 min, reaching a maximum of V15.5-fold above baseline
(n=4) at 10 min after hyperosmotic stimulation, and subse-
quently declining (Fig. 1D). Such a result indicates that a
dephosphorylation mechanism might exist for control of the
KS phosphorylation state. Our group is presently searching
for phosphatases that might potentially ful¢ll such a role. It
is interesting to note that time-dependent translocation of KS
oligomers from the plasma membrane to light vesicles via
acetylcholine stimulation has been reported . It remains
possible that the KS phosphorylation state a¡ects the intra-
cellular localization of KS.
3.3. Pyk2/RAFTK induces tyrosine phosphorylation of KS via
It has been suggested that one autophosphorylation site of
Pyk2/RAFTK, Y402, recruits c-Src to allow phosphorylation
of substrates [9,13]. Alternatively, the binding of c-Src to
Y402 of Pyk2/RAFTK might also lead to enhancement of
Pyk2/RAFTK kinase activity. To characterize the role of
Pyk2/RAFTK in the KS phosphorylation pathway in response
to osmotic stress, the e¡ects of expression of dominant-neg-
ative Pyk2/RAFTK mutants on tyrosine phosphorylation
were examined. COS7 cells were cotransfected with expression
vectors for HA-KS and FLAG-tagged wild-type Pyk2/
(Y402F), or the kinase-inactive Pyk2/RAFTK (K457R). After
site mutant Pyk2/RAFTK
Fig. 2. Osmotic stress-induced in vivo phosphorylation of KS is de-
pendent on Pyk2/RAFTK kinase activity. COS7 cells cotransfected
withexpression vectors forHA-KS and
(Pyk2) (WT, Y402F or K457R) were starved and treated with
300 mM D-sorbitol for 10 min at 37‡C. Cells were lysed and immu-
noprecipitated with anti-FLAG or anti-HA antibodies and analyzed
by immunoblotting with anti-FLAG, anti-KS, or anti-P-Tyr antibod-
ies. Densitometric analysis of KS phosphorylation is displayed in the
lower panel. Results are means?S.E.M. for four independent ex-
T. Nakamura et al./FEBS Letters 521 (2002) 190^194
the lysate protein contents were normalized, the cell lysate was
divided into three aliquots and immunoprecipitated with anti-
FLAG, anti-c-Src, or anti-HA antibodies. Osmotic stress-de-
pendent phosphorylation of KS was markedly reduced by ex-
pression of Y402F Pyk2/RAFTK from 16.2 to 2.6-fold (n=4)
compared with baseline measurements with cells expressing
wild-type Pyk2/RAFTK (Fig. 2). In cells expressing Y402F
Pyk2/RAFTK, c-Src was not observed to be su⁄ciently phos-
phorylated in response to osmotic stress (Fig. 2). In addition,
the e¡ects of the Src-family kinase inhibitor, PP2, on
RAFTK-mediated phosphorylation of KS were subsequently
examined. COS7 cells were treated with PP2 at various con-
centrations for 1 h, stimulated with sorbitol and compared
with PP2-untreated, sorbitol-stimulated controls. The osmotic
stress-dependent phosphorylation of KS was signi¢cantly de-
creased by addition of PP2 in a dose-dependent manner (Fig.
3), suggesting that KS phosphorylation is primarily achieved
with Src-family kinases. The results of the present work sug-
gest that activated Pyk2/RAFTK recruits Src-family kinases,
which could include c-Src, to phosphorylate tyrosine residue
125 of KS. Such ¢ndings indicate that Pyk2/RAFTK lies up-
stream of Src-family kinases in the signaling cascade by which
osmotic stress induces tyrosine phosphorylation of KS.
It is also of note that osmotic stress-dependent phosphory-
lation of KS was completely abolished by expression of
K457R Pyk2/RAFTK (Fig. 2). Moreover, c-Src was not phos-
phorylated in response to osmotic stress in the presence of
K457R Pyk2/RAFTK. Such results suggest that Pyk2/
RAFTK kinase activity is essential for osmotic stress-depen-
dent phosphorylation of KS. It is of interest that such results
also suggest the existence of a Src-family kinase-independent
KS phosphorylation pathway that is mediated via Pyk2/
RAFTK activation. One possibility is that Pyk2/RAFTK di-
rectly phosphorylates KS to a very low extent. Nonetheless,
Pyk2/RAFTK-mediated phosphorylation of KS is primarily
(V85%, Fig. 2) performed via Src-family kinases.
Pyk2/RAFTK is activated by a variety of stimuli that in-
crease the intracellular Ca2þlevel . The e¡ect of Ca2þon
osmotic stress-dependent phosphorylation of KS was further
investigated by eliminating extracellular calcium with calcium
chelators. The osmotic stress-dependent phosphorylation of
KS was signi¢cantly decreased by addition of EGTA (2 mM,
5 mM) or BAPTA-AM (5 WM, 10 WM) in a dose-dependent
manner, suggesting that Pyk2/RAFTK-mediated phosphory-
lation of KS was also dependent on extracellular calcium (Fig.
3). It is well established that osmotic stress a¡ects intracellular
free Ca2þconcentration . It should be noted that MAPK
pathways lie downstream of Pyk2/RAFTK-related signal
transduction ; KS is known to interact with MAPK and
a¡ect its activity [16,17]. The present study demonstrates that
extracellular Ca2þis necessary for both activation of Pyk2/
RAFTK and phosphorylation of KS following osmotic
Recently, post-translational modi¢cations of KS have been
reported. Tyrosine nitration of KS by oxidative stress facili-
tates oligomer formation in both in vitro [18,19] and in vivo
 experimentation. Furthermore, it is of note that KS pro-
tein in Lewy bodies is known to be nitrated . Moreover,
the Y125 residue of KS plays a critical role for oligomer for-
mation following nitrative stress . The present work has
demonstrated that Pyk2/RAFTK is involved in phosphoryla-
tion of the Y125 residue of KS via Src-family kinases. Such a
¢nding suggests that both extracellular signaling molecules
and stresses not only activate Pyk2/RAFTK, but also regulate
the phosphorylation state of KS.
Acknowledgements: We thank Y. Furuno for her technical support.
We also thank C.J. Hurt from the Johns Hopkins University School
of Medicine for manuscript corrections. This work was supported by
grants-in-aid for the Encouragement of Young Scientists (2001, 2002)
and for Scienti¢c Research on Priority Areas (C)-Advanced Brain
Science Project (2000, 2001) from the Japanese Ministry of Education,
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